Vibrio Cholerae From The Environment: Detection, Isolation, and Identification of

UNIT 6A.
5 Detection, Isolation, and Identication of

Vibrio cholerae from the Environment
Anwar Huq,
1
Bradd J. Haley,
1
Elisa Taviani,
1
Arlene Chen,
1
Nur A. Hasan,
1
and Rita R. Colwell
1,2
1
Maryland Pathogen Research Institute, Department of Cell Biology and Molecular
Genetics, University of Maryland, College Park, College Park, Maryland
2
University of Maryland Institute of Advanced Computer Studies, University of Maryland,
College Park, Maryland, and Bloomberg School of Public Health, Johns Hopkins University,
Baltimore, Maryland
ABSTRACT
Recent molecular advances in microbiology have greatly improved the detection of bac-
terial pathogens in the environment. These improvements and a downward trend in the
cost of molecular detection methods have contributed to increased frequency of detection
of pathogenic microorganisms where traditional culture-based detection methods have
failed. Culture methods also have been greatly improved, and the conuence of the two
suites of methods provides a powerful tool for detection, isolation, and characteriza-
tion of pathogens. While molecular detection provides data on the presence and type of
pathogens, culturing methods allow a researcher to preserve the organism of interest for
-omics studies, such as genomic, metabolomic, secretomic, and transcriptomic analy-
sis, which are rapidly becoming more affordable. This has yielded a clearer understanding
of the ecology and epidemiology of microorganisms that cause disease. In this unit, we
present commonly accepted methods for isolation, detection, and characterization of
V. cholerae, providing more extensive knowledge of the ecology and epidemiology of
this organism. This unit has been fully revised and updated from the earlier version with
the latest knowledge and additional information not previously included. Curr. Protoc.
Microbiol. 26:6A.5.1-6A.5.51.
C 2012 by John Wiley & Sons, Inc.
Keywords: Vibrio cholera
r
isolation
r
identication
r
detection
r
characterization
INTRODUCTION
It is well established that Vibrio cholerae, the causative agent of cholera, is autochthonous
to the aquatic environment globally and is not conned to cholera endemic areas (Kenyon
et al., 1984; Louis et al., 2003; Schuster et al., 2011). Further, V. cholerae cells encoding
genes and mobile elements known to cause disease in humans and confer resistance
to antibiotics are found in the absence of cholera. When appropriate methods are used,
V. cholerae can be detected in these aquatic environments throughout the year. V. cholerae
culturenegative water samples do not denitively indicate absence of this organism, as
cells can enter into a viable but nonculturable (VBNC) state in which they may not
form colonies on traditional bacteriological culture plates (Xu et al., 1982; Roszak and
Colwell, 1987). In the VBNC state, cell densities may be extremely low; therefore, if a
small amount of water sample is collected, it may not contain a sufcient number of cells
for detection. Thus, it is important that appropriate volumes of water be examined, using
appropriate methods to determine the presence of V. cholerae in a given sample.
This unit describes several widely accepted and highly cited methods used to detect,
cultivate, enumerate, and characterize V. cholerae cells in environmental samples, such
as water, sediment, plankton, and seafood, e.g., oysters. In developing countries, expo-
sure to V. cholerae typically occurs via consumption of natural surface water containing
Current Protocols in Microbiology 6A.5.1-6A.5.51, August 2012
Published online August 2012 in Wiley Online Library (wileyonlinelibrary.com).
DOI: 10.1002/9780471729259.mc06a05s26
Copyright
C 2012 John Wiley & Sons, Inc.
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6A.5.1
Supplement 26
Detection of Vibrio
cholerae from the
Environment
6A.5.2
Supplement 26 Current Protocols in Microbiology
Table 6A.5.1 Overview of Methods Presented in this Unit for Isolation and Detection of V. cholerae
Protocol Included methodology Protocol ID
Isolation of presumptive V. cholerae and
conrmation, using traditional methods
Specimen collection and transportation. Basic Protocol 1
Conventional bacteriological culture method for
V. cholerae
Basic Protocol 2
Alternate method for presumptive identication Alternate Protocol 1
Serogroup determination Basic Protocol 3
Molecular methods for detection of
V. cholerae Isolates
Preparation of DNA templates and conrmation
of suspected or presumptive V. cholerae by PCR
Support Protocol 1 and
Basic Protocol 4
Real-time PCR Basic Protocols 6 and 7
Colony blot hybridization with labeled DNA or
RNA hybridization probes
Basic Protocol 9
Molecular methods for direct detection of
V. cholerae in environmental samples
Fluorescence in situ hybridization (FISH) Basic Protocol 10
Direct PCR Basic Protocol 8
Direct detection and/or enumeration of
V. cholerae using immunological methods
Direct uorescent antibodydirect viable count
(DFA-DVC)
Basic Protocol 11
Indirect uorescent antibody (IFA) method Basic Protocol 12
V. cholerae in sufcient numbers to cause disease, whereas in developed countries, ex-
posure to V. cholerae often occurs via consumption of raw or undercooked shellsh or
travel to regions where cholera outbreaks occur frequently. Recent outbreaks of shellsh-
related vibrioses and cholera in developed countries demonstrate the need to dene a
reliable method for detection of these organisms in any environmentally derived food
source (Onifade et al., 2011). Both plankton and sediment play an important role in
the occurrence and survival of V. cholerae in water and oysters. Sediment and plank-
ton both can occur suspended within the water column. Sediment is known to harbor
V. cholerae (due to its polar electrical charge). Plankton are known to be natural reser-
voirs of V. cholerae, and zooplankton blooms facilitate increases in V. cholerae density.
Furthermore, an infectious dose of V. cholerae (6 10
6
cells; Cash et al., 1974) can be
present on a single plankton body (Huq et al., 1984; Rawlings et al., 2007). Presence of
V. cholerae in water, sediment, and plankton will also result in the presence of V. cholerae
in shellsh, as these organisms lter feed all three through their bodies and concentrate
high numbers of V. cholerae. It is, therefore, important to consider the type of sample
and desired outcome (detection, isolation, or enumeration) when choosing the appro-
priate bacteriological/molecular methods discussed here. For the readers convenience,
Table 6A.5.1 and Figure 6A.5.1 provide an overview of the methods provided in this
unit.
STRATEGIC PLANNING
As stated above, the type of sample and the desired outcome are integral in determining
which method(s) should be used to achieve satisfactory results. Furthermore, the skill
set of the laboratory members and laboratory resources are all essential in determining
which tests can be conducted, as all of these facets can vary dramatically from laboratory
to laboratory as well as over time within one laboratory.
Traditional culturing methods, although yielding only a small subset of culturable bacteria
in the original sample (<0.1%of total bacteria), do allowresearchers to archive individual
strains and run a variety of informative bench-top studies at a later date. These include
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6A.5.3
Current Protocols in Microbiology Supplement 26
collect samples
(Basic Protocol 1)
conventional
bacteriological culture
method for V. cholerae
(Basic Protocol 2)
serogroup
determination
(Basic Protocol 3)
preparation of DNA template
and confirmation of suspected
or presumptive V. cholerae
by PCR (Support Protocol
1, Basic Protocol 4, and
Tables 6A.5.3 and 6A.5.4)
or
preparation of DNA template
and real-time PCR
(Support Protocol 1 and Basic
Protocols 6 or 7)
alternative method for
presumptive identification
(Alternate Protocol 1)
colony blot hybridization
with labeled DNA or RNA
hybridization probes
(Basic Protocol 9)
direct PCR
(Basic Protocol 8)
direct fluorescent
antibody direct
viable count
(Basic Protocol 10)
indirect fluorescent
antibody method
(Basic Protocol 12)
fluorescence in situ
hybridization FISH
(Basic Protocol 10)
Figure 6A.5.1 Flowchart of methods (protocols) used to detect and/or isolate and characterize V. cholerae from the
environment.
genetic characterization by PCR, reconstruction of metabolic pathways by phenotypic
analyses, gene expression assays, and whole genome sequencing, among many others.
However, as stated earlier, reliance on traditional culturing methods only focuses ones
scope on those bacteria that are culturable while inherently ignoring those bacterial cells
that cannot grow on media but which do remain a signicant public health threat, as they
are capable of causing disease (Colwell, 1991).
To evaluate the presence of these organisms regardless of their culturability, a direct
detection method should be employed. Two microscopy methods are reliable, notably
uorescent antibody coupled with direct viable count (FA-DVC), or uorescence in situ
hybridization (FISH), a nucleic acid markerbased method of detection. FISH can be
utilized to detect genomic DNAsequences specic to V. cholerae regardless of serogroup,
while FA-DVC can be used to detect V. cholerae of serogroups O1 and O139, serogroups
associated with pandemic cholera. The direct uorescence antibody (DFA) detection of
serogroups O1 and O139 is more reliable than attempting to detect these strains via
culture methods, as targeting one serogroup in the environment is rather difcult to
do, considering the vast serodiversity of V. cholerae in the environment. Both methods
allow the researcher to quantify the target if no pre-enrichment step is used. Subsequent
bacterial isolation is not possible when using this method unless a parallel sample is
collected and processed using traditional culturing methods.
Direct molecular detection using the polymerase chain reaction (PCR) can also be em-
ployed to evaluate the presence of these organisms in environmental samples. These
methods rely on detection of species-specic nucleotide sequences, followed by ampli-
cation of the targeted nucleic acid and visualization on an agarose gel (conventional
PCR) or in an amplication plot using computer software (real-time PCR). Direct de-
tection using these methods can be applied directly to environmental samples, as well as
to samples following a pre-enrichment step in alkaline peptone water (APW; see recipe
in Reagents and Solutions) or estuarine peptone water (EPW; autoclaved APW with 1%
Detection of Vibrio
cholerae from the
Environment
6A.5.4
Table 6A.5.2 V. cholerae Positive Control Strains
Strain Serogroup Serotype Biotype Source
Year of
Isolation
V. cholerae NCTC 8457 O1 Inaba El Tor Clinical case (pilgrim en route to
Mecca) from a quarantine station in
El Tor (Al-Tur), Egypt
1930
V. cholerae MAK 757 O1 Ogawa El Tor Clinical case from Makassar,
Celebes Islands, a coastal region
(now Sulawesi, Indonesia)
1937
V. cholerae N16961 O1 Inaba El Tor Clinical case from Bangladesh 1975
V. cholerae 2010EL-1786 O1 Ogawa El Tor Clinical case from Artibonite
Department, Haiti
2010
V. cholerae NCTC 569B O1 Inaba Classical Clinical case in India 1948
V. cholerae O395 O1 Ogawa Classical Clinical case from India 1964
V. cholerae LMG 4406 non-O1/non-
O139
NA Albensis Fish isolate from the Elbe River,
Germany
1896
V. cholerae 26 non-O1/non-
O139
NA NA Environmental isolate from Santa
Cruz, CA (Pacic Ocean)
1983
V. cholerae MO45 O139 NA NA Clinical isolate from Madras, India 1992
peptone added). All of these methods are highly sensitive, but it must be stated that
absence of evidence of V. cholerae by a particular method does not conrm absence
of this organism. As with all microbiological and molecular methods, inhibitors of the
polymerase chain reaction, as well as quality and quantity of the collected sample and
sensitivity of detection for each method, must be taken into account when interpreting
results. Replicate sample collection and replicate laboratory analyses further strengthen
the statistical signicance of results, but can dramatically increase level of effort as well
as nancial commitment.
Selection of sampling sites and specimen collection should be based on the goals of the
study. It is valuable to select a variety of sites that are diverse in their ecology, with some
sites selected with the a priori notion that they will harbor high densities of V. cholerae
and others selected with the notion that they will harbor low densities of V. cholerae.
If it is the goal to evaluate the presence or densities of V. cholerae in oysters, then
the overlying water column, underlying sediment, and planktonic organisms within the
water column should also collected to discern the ecology of V. cholerae near the oyster
beds of interest. During specimen collection, environmental parameters, such as, water
temperature, salinity, conductivity, turbidity, and pH, should be recorded. For ecological
investigations of organisms autochthonous to the aquatic environment, it is necessary to
collect and process water, sediment, plankton, and, if possible, seafood (oyster) samples
in a time series, preferably over a multi-year period with the aim to conduct sampling at
regularly spaced intervals.
The collection of data over longer study periods allows researchers to estimate those pa-
rameters that may be associated with changes in V. cholerae densities or presence/absence
in the selected area of interest. For studies utilizing these data to develop predictive mod-
els of V. cholerae in water bodies, readers are encouraged to review the reports of
Louis et al. (2003), Huq et al. (2005), Constantin de Magny et al. (2008), and Turner
et al. (2009). In addition to consideration of time, resources, and logistics, the proto-
cols in this unit allow a researcher the option to isolate and/or detect toxigenic Vibrio
cholerae or total Vibrio cholerae. For all assays, media, materials, and methods should be
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6A.5.5
validated with a positive control and a negative control. Several commonly used
V. cholerae positive-control strains that are typically available from culture collections,
such as the American Type Culture Collection (ATCC), are listed in Table 6A.5.2.
CAUTION: V. cholerae is a Biosafety Level 2 (BSL-2) pathogen. Follow all appropriate
guidelines and regulations for the use and handling of pathogenic microorganisms. See
UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more information.
ISOLATION AND IDENTIFICATION OF V. CHOLERAE USING
TRADITIONAL METHODS
Although developed several decades ago, traditional culturing methods continue to be im-
proved, with the result of more reliable detection of V. cholerae from environmental sam-
ples. The process involves either directly plating the study sample (water, plankton, sedi-
ment, shellsh) onto TCBS, TTGA, or CHROMagar Vibrio (http://www.chromagar.com),
or pre-enrichment of the sample in alkaline peptone water followed by streaking for iso-
lation onto one or a combination of these three agars. For environmental samples, it
is highly recommended to use a pre-enrichment step to improve detection or isolation.
Following an incubation period, presumptive V. cholerae can be isolated from these me-
dia and conrmed either by biochemical analyses (Huq et al., 2006) or immediately by
PCR. Isolates can then be serogrouped as O1, O139, or non-O1/non-O139 by a slide
agglutination assay using antisera for the O1 and O139 antigens or by PCRusing primers
developed to target O1 and O139 coding regions of the genomic DNA. These V. cholerae
isolates can then be archived with replicates in nutrient agar containing 0.5% NaCl over-
laid with sterile mineral oil or as a glycerol stock at 70
C. Stock cultures of V. cholerae

should be periodically re-streaked for isolation to ensure their viability and purity.
BASIC
PROTOCOL 1
Specimen Collection and Transportation
If the aim is to detect and isolate V. cholerae in water samples, then screening of concen-
trated water samples and plankton samples and plankton-free water is recommended,
since a combination of all types of samples provides a higher probability of V. cholerae
detection as well as a better understanding of the role of plankton in V. cholerae dynamics
in the study region (Huq et al., 1990; Binsztein et al., 2004; Huq et al., 2005; Turner
et al., 2009; Lizarraga-Partida et al., in preparation). Water collection bottles should be
cleaned with detergent; however, the detergent must not leave residue and should not
be antibacterial. The bottles should be pre-sterilized in an autoclave for 15 to 20 min at
121
C prior to use. Polypropylene bottles should be used for water samples and glass
bottles should be used for plankton samples. A sufcient volume of water and plank-
ton should be collected to ensure that appropriate analyses can be performed. Plankton
samples should be collected by passing water through a 200, 64, and/or 20-m pore size
plankton net by towing the net manually or behind a boat. Alternatively, a known volume
of water can be manually passed through the net by bucket pouring or pumping, which
can be useful for quantitative analysis (Huq et al., 2005).
Processing of samples should begin soon after collection (typically within 24 hr of col-
lection). If enumeration of V. cholerae is desired, then the sample should be stored in a
cool box at a temperature of 10
to 15
C until processing begins (not to exceed 8 hr;

Clesceri et al., 1998). However, a recent study has demonstrated that transporting samples
at ambient air temperature prior to processing can enhance the culturability of V. cholerae
in the sample (Alam et al., 2006a,b). Based on type of examination, samples may require
treatments, such as addition of direct viable count (DVC) reagents, before proceeding
with further examination and testing. It is recommended that basic physiochemical pa-
rameters, e.g., temperature, salinity, pH, dissolved oxygen, and conductivity of the water
be measured on site at the time of collection, as it is known that V. cholerae densities
can be inuenced by such parameters. If the study site is inuenced by tides, coasts, and
Detection of Vibrio
cholerae from the
Environment
6A.5.6
estuaries, for example, the prevailing tidal level at the time of sampling should be
recorded. Furthermore, in addition to water temperature, signicant correlation of water
depth, rainfall, conductivity, and copepod density with cholera outbreaks has been re-
ported, with lag periods from 0 to 8 weeks from optimum environmental conditions to
cholera outbreaks (Huq et al., 2005). These parameters can be measured on site using
portable meters (such as HACH Model CO150 conductivity meter, HACH Chemical
Company, or dissolved oxygen and pH meter Model 210A, Orion Laboratories). It is
important to note that in all studies of the ecology of microorganisms in the environment,
several rounds of practice sampling and processing should be considered to optimize
the volumes of water, plankton, or other biotic and abiotic media to be collected in
order to detect or recover the specic microorganism of interest, as well as to identify
missing equipment and reagents and evaluate the ability to comprehend and adhere to
the standard methods described herein. It is hard to keep the sample collection volumes
constant, as the amounts (volumes) may need to be adjusted depending on the quality of
water due to presence of seasonal plankton. The authors of this unit strongly encourage
investigators to use several methods and several types of media throughout the duration
of their studies. All methods have some error or detection limits associated with them
and, therefore, use of multiple approaches will allow investigators to converge on the
most accurate answer. Water, sediment, and oysters isolated fromdifferent environments,
both on a global and local scale, may have different chemistry associated with them, and,
therefore, the different media requirements discussed in this unit may perform more or
less effectively over time and space. Diversifying ones approach to gathering data on the
ecology of V. cholerae will help overcome these challenges. The authors also encourage
the investigators to use a range of incubation temperatures for APW enrichment and
growth on selective agar plates. It is well known that V. cholerae can grow within a
wide range of temperatures, and that this group of organisms is phenotypically heteroge-
neous. Therefore, utilizing two incubation temperatures may increase the probability that
V. cholerae is detected and/or isolated. It should be known that this may effectively double
the amount of work downstream, but the investigators should also be aware that several
methods of isolation and detection will allow convergence on an accurate analysis.
Materials
Phosphate-buffered saline (PBS; APPENDIX 2A), sterile
Pre-sterilized 500-ml glass (Qorpak) and 1000-ml polypropylene containers
(Nalgene)
Simple plankton nets, 200-m, 64-m, and 20- m, or nets of different mesh sizes
(for size ltration of plankton); Aquatic Research Instruments
(http://www.aquaticresearch.com/) or SEA-GEAR (http://www.sea-gear.net/);
see Fig. 6A.5.2)
Portable meter(s) that measure temperature, dissolved oxygen, pH, turbidity, and
salinity (HACH Company; Geo Scientic; YSI/Xylem); turbidity and salinity
can be measured ex situ, i.e., in the laboratory, preferably immediately after
sample collection.
Bucket of known volume: .g., 4 or 5 liters (optionally used for pouring water
through plankton nets)
Piston corer (Aquatic Research Instruments; http://www.aquaticresearch.com/) or a
Peterson grab (Wildlife Supply Company; http://www.wildco.com/)
Wide-bore serological pipets
Oyster tongs or dredge
92-oz. Whirl-Pak Bags (eNasco; http://www.enasco.com/)
Autoclaved and pre-weighed 100-ml polypropylene container (optionally used
when collecting sediment from an oyster bed)
Scooper/spoon that has been wrapped in aluminum foil and autoclaved (optionally
used when collecting sediment from an oyster bed)
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6A.5.7
To collect water sample
1a. Uncap pre-sterilized plastic bottle, submerge bottle to ll. Fill to half of volume of
bottle, re-cap, shake to rinse, and discard. Repeat 3 to 5 times. Rinse downstream of
water sample collection site if possible.
2a. Remove the sample bottle from the water. Cap, leaving enough air in the bottle for
agitation and mixing.
To collect plankton
1b. Rinse plankton net and cod-end of the net (see Fig. 6A.5.2) in the body of water to
be sampled (downstream of sample collection site if possible).
2b. Filter 10 to 100 liters water through the plankton net by towing, using a calibrated,
net-mounted owmeter, or by pouring known volumes through the net with a smaller
bucket (the bucket should be clean, but without detergent, and rinsed several times
with sample water, downstream of the sample collection site, immediately prior to
sample collection).
3b. For each plankton fraction of interest, rinse inside the net of the plankton net,
followed by the cod-end, with sterile PBS using a spray or squirt bottle, or with
excess plankton-free water collected after pouring sample water through the net.
This allows any plankton adhering to the net to be ushed down to the cod-end for
collection.
4b. Remove cod-end fromthe plankton net and decrease volume to 100 ml by continuing
to lter.
This can be done by gently swirling the cod end so that water passes through the mesh
siding of that cod end.
5b. Measure (with a sterile graduated cylinder) and decant plankton fraction into a sterile
glass bottle, making sure to label that bottle with the collected plankton fraction size.
6b. Repeat this process for all plankton fractions of interest.
7b. Finally, collect 500 ml to 1000 ml water that passes through the 20-m plankton net.
This is plankton-free water.
To collect sediment sample
1c. Collect sediment with a piston corer or a Peterson grab.
2c. Sample a sediment-water interface with a wide-bore serological pipet and put in
a sterile bottle (this sediment-water interface sample can be treated as a separate
cod-end metal ring
three-point
bridle
Figure 6A.5.2 Simple plankton net (Aquatic Research Instruments). The net is composed of
a metal ring and bridle, a heavy-duty nylon net of variable mesh size, and a PVC cod-end or
collecting bucket which can be removed for easy sample collection. A ow meter may be mounted
in the mouth of the net to the metal ring to measure volume when the net is towed.
Detection of Vibrio
cholerae from the
Environment
6A.5.8
sediment sample and processed alongside the other media collected, following the
same protocols).
3c. Remove the remaining sediment from the sampling apparatus aseptically and put
into a sterile bottle.
To collect shellsh
Shellsh collection in many areas is regulated by local or state governments. It is imper-
ative to check these regulations with your local Fish and Wildlife department or other
relevant government bodies prior to sample collection. Collection of shellsh may re-
quire purchase of a permit and, in some locations, may be done only on certain days. The
oyster processing protocols described here can be used to evaluate store-bought oysters.
However, these oysters may have been depurated and stored for some time, and results
from these analyses should be described with respect to post-harvesting conditions, not
the environment from which these oysters were harvested. Additionally, since oyster
shells may be sharp, it is advised to wear gloves whenever handling oysters in order to
avoid injury.
1d. Collect oysters from boats by using oyster tongs or a dredge or hand collect at low
tide when the oyster beds are easily accessible.
2d. Examine oysters for viability in the eld, keeping only those oysters that are tightly
closed. Rinse sediment off of the oyster shells with surface water, but do this only
after water and plankton samples have been collected.
Tapping the mid-shell point of an oyster on a hard surface will allow determination of
whether oyster meat is present in the oyster shell, because a hollow sound will be heard
when an empty oyster is tapped. Rinse sediment off of the oyster shells with surface water,
but do this only after water and plankton samples have been collected.
3d. Place viable oysters in ventilated 92-oz. Whirl-Pak Bags and transport in a cool
container, but not immediately next to ice or cool packs. Do not re-immerse oysters
in water after collection.
To collect sediment from an oyster bed
If oysters are already being collected during sample collection, it is possible to collect
sediment samples at the same time in order to avoid using a piston corer, as direct
sediment sample collection from a hard oyster bed (also known as an oyster reef) may
be difcult. When determining whether or not oysters are present in the shell by tapping,
hollow oyster shells may contain sediment inside; therefore it is imperative to open the
oyster shells to check for sediment. If sediment is found, use a scooper that has been
previously wrapped in aluminum foil and autoclaved to scrape the sediment off the shell.
Place sediment in a sterile, pre-weighed 100-ml polypropylene container.
Transportation and/or processing
Transport samples to the laboratory for processing or, preferably, begin processing on
site within 1 hr of collection. For samples that require transport to the laboratory to be
processed after collection, store and/or transport the samples in a cold box at 10
to
15
C. Alternatively, samples can be kept in the dark at ambient air temperature (22
to
25
C) after collection for up to 24 hr, as this may enhance recovery of Vibrio species as
shown in a recent study (Alam et al., 2006a,b), and can be a useful approach for detecting
vibrios in those environments where the organism is predominantly in the viable but
non-culturable (VBNC) state (Alam et al., 2006a,b). This aspect of the protocol may
need to be optimized for the water source and environmental conditions.
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6A.5.9
BASIC
PROTOCOL 2
Conventional Bacteriological Culture Method
Conventional culture methods for isolating V. cholerae fromenvironmental water samples
rely on an enrichment step(s) in broth and plating on selective media, followed by conr-
mation using a series of biochemical tests or PCRand serological tests to determine strain
serotype. Alternatively, biochemical tests can be bypassed, and conrmation can be done
using PCR if adequate supplies of PCR reagents are available. Most water and plankton
samples require some amount of concentration. Running several practice rounds at the
desired study site will help estimate appropriate volumes of sample that should processed
in order to achieve desired goals. However, it should be pointed out that environmental
V. cholerae densities are not static, so volumes deemed to be appropriate at one sampling
point may not yield the same results at another time. We address this inconsistency by
urging the investigator to use multiple methods (if resources are available) to isolate,
detect, and characterize environmental V. cholerae during the course of the study. Water
samples, both whole water and plankton-free water, should be concentrated by ltration
using 0.2-m polycarbonate membrane lters. A good starting volume is 500 ml; 1000
ml can be used if water passes easily through the polycarbonate membrane. If the 0.2-m
polycarbonate membrane lter clogs quickly, multiple lters can be used per sample. It
is preferable to use 0.2-m pore size lters as opposed to 0.45-m pore-size lters, as
VBNC bacteria are often <0.45-m in size. Polycarbonate membranes are preferable to
nitrocellulose membranes, as the cells sit on top of the polycarbonate lters and are easily
removed by vortexing, while cells get caught in the bers of nitrocellulose membranes.
It is imperative that the cells be physically agitated off of the membrane so that they may
migrate to the microaerophilic layer of the enrichment broth discussed below.
Overnight enrichment is performed using alkaline peptone water (APW), pH 8.6, and
some investigators recommend two successive enrichments. Surface aliquots from the
microaerophilic pellicle layer are streaked for isolation onto selective bacteriological me-
dia. Three commonly used selective media for V. cholerae isolation are thiosulfate citrate
bile-salts sucrose (TCBS) agar, tellurite taurocholate gelatin agar (TTGA), also known as
Monsur medium(Monsur, 1961), and CHROMagar Vibrio (http://www.chromagar.com/).
V. cholerae appears as translucent, at, yellowcolonies with elevated centers on TCBS; as
colorless colonies on TTGA, often with a characteristic dark center after 2 days growth,
surrounded by a halo, which appears due to the hydrolysis of gelatin; and as turquoise
colonies on CHROMagar Vibrio (Fig. 6A.5.3). If possible, use more than one medium,
as that may allow convergence on the best results. APW enriches for many bacteria other
than V. cholerae; therefore, direct plating of samples onto TCBS, TTGA, and/or CHRO-
Magar Vibrio can be done in parallel with APW enrichment, and may yield V. cholerae
colonies that can be used for further analyses. This direct plating method is benecial in
instances when overgrowth of non-target bacteria occurs on solid media after overgrowth
in APW.
TCBS is a highly selective medium that may, in rare instances, inhibit growth of
V. cholerae and at the same time allow growth of non-Vibrio organisms, such as
Aeromonas sp., Staphylococcus sp., and Shewanella sp. TCBS is commercially avail-
able and produced by several companies that have global distribution. TTGA medium
is considered to be less inhibitory to V. cholerae cells; however longer incubation times
are needed (48 hr) to observe the dark center that is characteristic of V. cholerae on
this medium. It may be difcult to distinguish V. cholerae from V. parahaemolyticus on
this medium; therefore, modied TTGA medium can be used in which V. cholerae
colonies appear as brilliant blue with uorescence in 24 hr or less, by adding -
galactosidase (4-methylumbelliferyl---galactosidase) (OBrien and Colwell, 1985).
CHROMagar Vibrio, primarily used for the detection of V. cholerae, V. parahaemolyticus,
V. vulnicus, and V. alginolyticus, distinguishes these organisms from each other based
on a proprietary chromogenic reaction that precludes making it in the laboratory from
Detection of Vibrio
cholerae from the
Environment
6A.5.10
A
B
C
Figure 6A.5.3 Growth of V. cholerae O1 on (A) TCBS, (B) TTGA, (C) CHROMagar Vibrio,
courtesy of Dr. Munir Alam, International Center for diarrheal Disease Research, Bangladesh.
scratch. TCBS and CHROMagarVibrio need not be autoclaved, and incubation times for
these media are shorter (24 hr) than for TTGA. TTGA medium must be autoclaved prior
to use. Once presumptive strains are puried on a nonselective medium, such as gelatin
agar, modied nutrient agar, or Luria Bertani (LB) agar with 1% NaCl, they are con-
rmed and serogrouped by PCR or by simple slide agglutination, employing polyclonal
V. cholerae O1, O139 and monoclonal Inaba and Ogawa antiserum.
Materials
Water, plankton, oyster, or sediment samples (Basic Protocol 1)
10 and 1 alkaline peptone water (APW), pH 8.6 (see recipe)
Thiosulfate citrate bile-salts sucrose (TCBS) agar plates (see recipe)
Nonenteric
Gamma
Proteobacteria
6A.5.11
Tellurite taurocholate gelatin agar (TTGA) plates (see recipe)
CHROMagar Vibrio (CA; http://www.chromagar.com/)
Phosphate-buffered saline (PBS), pH 7.4 (APPENDIX 2A), sterile
95% alcohol for ame sterilization of forceps, loops, needle, bacterial cell spreader
Gelatin agar (GA; see recipe), modied nutrient agar (see recipe), or Luria-Bertani
agar (LB) plates (see APPENDIX 2C)
Oxidase reagent: 1% (w/v) N,N,N
-tetramethyl-p-phenylenediamine
dihydrochloride (e.g., Sigma)
Filter membranes, 47-mm diameter, 0.22-m pore size (Millipore)
Filter apparatus with vacuum source
Forceps
50-ml centrifuge tubes (e.g., BD Falcon)
Inoculating loops, needles, and cell spreaders (see UNIT 4A.1)
Tissue homogenizer (hand-held) to homogenize plankton (Kimble Chase Life
Science and Research Products)
Enrichment ask (sterile 150-ml Erlenmeyer asks)
Cut-resistant oyster shucking glove (Chef Revival, http://www.chefrevival.com/)
Oyster knife (Dexter-Russell; http://www.dexter1818.com/)
Autoclavable kitchen blender (Waring, 700G)
Filter paper
Sample processing
If not specied, assume APW is 1 APW.
1a. For plankton samples: Filter concentrated plankton sample through a 47-mm, 20-m
pore size polycarbonate lter (this step may take a while depending on the abundance
of plankton in the sampled water). Add the lter(s) with attached bacteria to a 50-ml
centrifuge tube with 25 ml of sterile PBS.
i. Vortex the centrifuge tube in order to remove cells from the lter.
ii. Add 1-ml whole plankton to 25 ml of 1 APW.
iii. Add 100 to 1000 l of whole plankton directly onto TCBS and/or CA and TTGA
plates and spread using a bacterial cell spreader.
iv. Homogenize 10-ml of concentrated plankton samples using a glass tissue grinder
by moving the pestle up and down in the tube, while rotating, 10 to 20 times. Add
1 ml homogenized plankton to an enrichment ask containing 25 ml APW.
v. Add 100 to 1000 l of homogenized plankton directly onto TCBS and/or CA and
TTGA plates and spread using a bacterial cell spreader.
1b. For water samples: Filter 100 to 1000 ml water (depending on bacterial density, see
Basic Protocol 1) through a 47-mm, 0.22-m pore size polycarbonate lter. Add the
lter(s) with attached bacteria to a 50-ml centrifuge tube with 12 ml of sterile PBS.
Vortex vigorously for 5 min to detach the bacteria from the lter.
i. Add 100 to 1000 l of the PBS containing detached bacterial cells directly onto
TCBS and/or CA and TTGA plates and spread until plates appear dry, using a
bacterial cell spreader.
ii. Add 1 ml PBScontaining detached bacterial cells to an enrichment ask containing
25 ml APW.
This step may be done in replicate to increase the probability of isolating V. cholerae.
However, it should be known that replication at this step dramatically increases the
magnitude of analyses downstream.
Large volumes or highly turbid sample water may require more than one polycarbonate
lter, as they will clog.
Detection of Vibrio
cholerae from the
Environment
6A.5.12
The remaining PBS containing detached bacterial cells can be stored at 20
C for other
direct molecular detection methods, including metagenomic analysis.
1c. For oyster samples: Rinse and scrub exterior of oysters in sterile deionized water,
making sure to remove sediment from the oyster hinge. With extreme caution,
wearing a cut-resistant oyster-shucking glove, shuck oysters with a sterile oyster
knife.
It may help to also hold the oysters level on a table with a towel between your glove and
the oyster. This may add protection and stability.
i. When shucking, with the curved-side of the knife facing downwards, trace the
knife along the upper shell, making sure to sever the adductor muscle which holds
the oyster shells shut. Shuck enough oysters to produce 250 g of oyster meat as
well as liquid surrounding the oyster meat and add to an autoclavable kitchen
blender.
ii. Add an equal volume of sterile PBS and homogenize for 90 sec.
iii. Add 20 ml of homogenized oyster sample to an enrichment ask containing 80 ml
of 10 APW.
iv. Add 100 to 1000 l of oyster homogenate to TCBS and/or CA and TTGA plates
and spread using a bacterial cell spreader.
This step may be further adjusted to include both direct plating of 100 to 1000 l onto
agar as well as plating serial dilutions of homogenized oyster meat in sterile PBS.
If an autoclavable blender is not available, the interior of the blender pitcher can be ster-
ilized with 10% bleach or 70% alcohol followed by multiple rinses with sterile deionized
water or PBS. It is imperative that all bleach or alcohol be removed prior to adding the
oyster meat. Alternatively, a Stomacher (http://www.seward.co.uk/) could be used.
To sterilize the oyster knife, dip blade into 95% ethanol and then pass through a ame.
If the oyster knife is autoclavable, it can be wrapped in aluminum foil and sterilized at
121
C for 15 min. Oysters can be shucked when the blade is cool.

1d. For sediment samples: Weigh sediment samples and add an equal volume of sterile
PBS based on the weight of the sediment and an assumed density of 1.0, and vortex
or shake well.
i. Add 100-ml sediment/PBS slurry to an enrichment ask containing 11 ml 10
APW.
ii. Additionally, add 100 to 1000-l sediment/PBS slurry directly onto TCBS and/or
CA and TTGA plates, and spread using a bacterial cell spreader.
This step may be further adjusted to include both direct plating of 100 to 1000 l onto
agar as well as plating serial dilutions of sediment in sterile PBS.
2. Incubate the APW enrichment asks statically for 16 hr at 30 to 35
C (overnight).
In all cases, APW enrichment asks should not be disturbed or agitated during or after
incubation, as Vibrio species tend to migrate to the liquid-air interface.
A range of incubation temperatures can be used, as V. cholerae is known to grow between
12
and 42
C. Lower incubation temperatures for longer periods of time may allow for
more V. cholerae cells to grow while also allowing for other competitive bacteria to grow
as well. Higher incubation temperatures may inhibit growth of some environmentally
stressed V. cholerae cells but will also inhibit growth of competitive bacteria species. It
may be benecial to consider testing two incubation temperatures and choosing which
yields better results for your sample area of interest. Alternatively, replicate asks can
be incubated at each temperature to increase the probability of isolating V. cholerae (see
italicized note immediately after step 1b, above).
Nonenteric
Gamma
Proteobacteria
6A.5.13
3. Incubate TCBS and CA plates at 30 to 35
C for 24 2 hr and TTGA plates at 30
to 35
C for 48 hr.
The same approach as stated under step 2 for incubation temperatures of APW inoculates
can be applied, and should be considered for incubation of agar plates.
4. Subculture smooth, at, sucrose-fermenting, yellow colonies from TCBS and/or
turquoise blue colonies from CA, and translucent, dark-centered colonies with halo
zones from TTGA (see Fig. 6A.5.3) onto LB, GA, and/or modied nutrient agar
plates.
Selective plating post-APW enrichment
5. After enrichment in APW, collect surface growth (which may be present as a whitish
lm) from the enrichment ask with an inoculating loop and streak onto TCBS
and/or CA and TTGA plates.
6. Incubate the plates 16 to 24 hr at 30
to 35
C for TCBS and CA or 48 hr at 30
to
35
C for TTGA.
7. Subculture smooth, at, sucrose-fermenting, yellow colonies from TCBS and/or
turquoise blue colonies from CA, and translucent, dark-centered colonies with halo
zones from TTGA (see Fig. 6A.5.3) onto LB, GA, and/or modied nutrient agar
plates, respectively.
To subculture presumptive V. cholerae colonies from these selective media, touch the
top-center of the colony using a sterile inoculating loop. Take care not to touch the
surface of the agar or any other surrounding bacteria (even if they too are presumptive
V. cholerae). If plates are heavily overgrown with bacteria, try to touch the top-center of
a presumptive V. cholerae colony and re-streak onto selective media to isolate a single
colony.
If TCBS is used, morphology should be registered only on recent and well isolated colonies.
Perform oxidase test
1% (w/v) tetramethyl-p-phenylenediamine dihydrochloride solution (oxidase reagent)
should be prepared the same day the test is being performed.
8. Moisten lter paper with 1% tetramethyl-p-phenylenediamine solution. Using a
platinum loop, pick colonies and spread them onto the moistened lter paper. Look
for a purple or violet color appearing in 10 sec, which indicates a positive read.
ALTERNATE
PROTOCOL 1
Bacteriological Culture Method for Situations of Limited Resources
This alternative method can be used to presumptively estimate the presence of culturable
V. cholerae in surface waters in regions where even general microbiology resources
are limited. Bacteriological methods are based on the work of Choopun et al. (2002).
Yellow colonies isolated from TCBS that give negative reactions (no color change)
in esculin hydrolysis and arginine dihydrolase assays can be presumptively identied
as V. cholerae.
Sterilization procedures
Glassware can be used for assays and can be sterilized by wrapping in aluminum foil
or newspaper and then heated in an oven at 180
C for 2 hr. Sterilization can also be

accomplished by microwaving materials for 5 min. Investigators must make sure that
no non-microwavable materials are used (e.g., aluminum foil). Mineral oil should be
sterilized by heating in an oven at 170
C for 1 to 2 hr. If it is not possible to procure

autoclavable sample-collection bottles, then store-bought drinking water bottles may be
used. It is important to rinse these bottles with surface water from the collection site three
to ve times prior to collecting the sample for analysis.
Detection of Vibrio
cholerae from the
Environment
6A.5.14
Materials
Water sample (Basic Protocol 1)
10 and 1 alkaline peptone water (APW), pH 8.6 (see recipe)
Thiosulfate citrate bile-salts sucrose (TCBS) agar plates (see recipe)
Tellurite taurocholate gelatin agar (TTGA) plates (see recipe)
CHROMagar Vibrio (CA; http://www.chromagar.com/)
Brain-heart infusion agar stabs (see recipe) containing 0.1% (w/v) esculin and
0.05% (w/v) ferric chloride
Luria-Bertani broth containing 1% (w/v) L-arginine (pH 6.8) and phenol red
powder added as an indicator
Sterile mineral oil
Inoculating loops and needles, sterile (see UNIT 4A.1)
1. Add 100-ml of water to be tested to a sterile bottle containing 900-ml 10APW and
shake or swirl well.
2. Incubate at 30
to 35
C overnight.
3. With a sterile loop or toothpick, dab the upper pellicle layer of the incubated sample
and streak for isolation onto a selective medium for V. cholerae isolation (TTGA,
TCBS, or CA). Incubate plates at 30
to 35
C overnight (or 48 hr if TTGA agar is

used).
If incubators are not available, then the 10 APW inoculation can be left to sit at room
temperature for 24 hr and agar plates can be inverted (agar-side up) and left to sit in the
dark until growth occurs (up to 2 days).
4. For esculin hydrolysis assay, gently touch the top and center of a yellow non-
swarming colony with a sterile loop or toothpick and inoculate into brain-heart
infusion agar stabs containing 0.1% esculin and 0.05% ferric chloride.
A non-swarming colony is one that is symmetrical and <3 mm in diameter.
5. For arginine dihydrolase assay, simultaneously inoculate Luria-Bertani broth con-
taining 1% (w/v) L-arginine (pH 6.8) and phenol red powder (Difco) added as an
indicator. After inoculation, cover with 0.5 cm sterile mineral oil.
6. Incubate esculin hydrolysis assays at 30
to 35
C for 72 hr, or at room temperature

for approximately 96 hr, and arginine dihydrolase assays for 24 hr at 35
C or 48 to
72 hr at room temperature.
Blackening of the esculin hydrolysis medium after incubation indicates that that there
was a positive reaction, while appearance of a red color after incubation of the arginine
dihydrolase assay is considered a positive reaction. For both assays, a negative control
(uninoculated assays) should be incubated at the same temperature for the same time,
as a comparator. Used materials, including inoculated media, can be sterilized by mi-
crowaving for 5 min. Used materials can also be sterilized in a dry oven at 180
C for
2 to 3 hr.
BASIC
PROTOCOL 3
Serogroup Determination
More than 200 serogroups of V. cholerae have been described to date based on anti-
genic properties of cell surface polysaccharides, of which serogroups O1 and O139 have
been implicated in epidemics of cholera while non-O1/non-O139 serogroups are known
to cause sporadic outbreaks. However, serogroup O37 was responsible for a localized
outbreak of cholera in Czechoslovakia and Sudan. The remaining serogroups, collec-
tively and commonly termed as non-O1/non-O139, predominate among the strains of
V. cholerae isolated from the aquatic environment (Sack et al., 2003). Although reported
Nonenteric
Gamma
Proteobacteria
6A.5.15
mostly in O1 and O139 serogroup clinical isolates, the cholera toxin gene can be found in
non-O1/non-O139 strains from the aquatic environment globally, as well as other Vibrio
species (Chakraborty et al., 2000; Malayil et al., 2010). However, because of the epidemic
potential, the method to determine O1 and O139 serogroup is mentioned below. Strains
other than O1 or O139 serogroup need not be serogrouped unless there is a special need.
In such cases, those strains should be sent to a Reference Center for serotyping, since
antisera for serogroups other than O1 and O139 are not commercially available.
Materials
Bacterial growth (6- to 16-hr subculture on nonselective medium; see Basic
Protocol 2)
Phosphate-buffered saline (PBS; APPENDIX 2A)
Antiserum for serogroup O1 and O139 V. cholerae
Glass slides
Wax pencils
1. Draw two squares approximately 6 cm 6 cm on a microscope slide with a wax
pencil. Add one drop of PBS in each square .
2. Add a loopful of fresh growth (6- to 16-hr subculture on non-selective media, e.g.,
LB, GA, or modied nutrient agar) into each drop and resuspend.
3. Add an equal-sized drop of group O1 polyvalent antiserum (undiluted) to one of the
drops.
4. Mix the antiserumculture suspension by tilting the slide back and forth. Determine
if the reaction clumps (i.e., agglutinates) within 0.5 to 1 min, indicating a positive
result.
Agglutination can be better visualized by looking through microscope slide with a bright
lamp in the near background.
Autoagglutination, clumping in the saline solution without antiserum, is indicative of a
rough morphotype and cannot be typed by antisera.
5. Test non-O1 serogroup colonies with O139 antisera, following steps 1 to 4, above,
replacing the O1 antiserum with O139 antiserum.
MOLECULAR METHODS FOR DETECTION AND IDENTIFICATION OF
V. CHOLERAE ISOLATES
Conventional PCRand real-time PCRhave recently been used to characterize V. cholerae
strains, along with conrming a strain as V. cholerae or conrming the presence of
V. cholerae in environmental or clinical samples. Further, the increase in genome
sequences now available has allowed development of PCR-based typing schemes
for analysis of variants of strains, genes, and mobile genetic elements, includ-
ing pathogenicity islands. The body of knowledge derived from the V. cholerae
pangenome continues to provide researchers with targets useful in strain identication and
characterization.
Identication and characterization of suspected or presumptive V. cholerae isolates
by PCR
The polymerase chain reaction (Kramer and Coen, 2001) is a useful alternative to labor-
intensive biochemical tests, which are occasionally difcult to interpret, often requiring
replication as well as several days of incubation and media preparation. Ideally, at least
an oxidase test (Basic Protocol 2) should be done on presumptive colonies to reduce
Detection of Vibrio
cholerae from the
Environment
6A.5.16
Table 6A.5.3 PCR Targets and Primers for V. cholerae
Target Primer Sequence (5
-3
) Amplicon Reference
ITS pVC-F2 TTAAGCSTTTTCRCTGAGAATG 295-310 Chun et al. (1999)
PVCM-R1 AGTCACTTAACCATACAACCCG
ctxA PCTA-94F CGGGCAGATTCTAGACCTCCTG 563 Fields et al. (1992)
PCTA-614R CGATGATCTTGGAGCATTCCCAC
toxR pToxR-101F CCTTCGATCCCCTAAGCAATAC 778 Rivera et al. (2001)
pToxR-837R AGGGTTAGCAACCGATGCGTAAG
tcpA pTcpA-72F CACGATAAGAAAACCGGTCAAGAG 452 Keasler and Hall (1993)
pTcpAET-477R CGAAAGCACCTTCTTTCACGTTG 621
pTcpACL-647R TTACCAAATGCAACGCCGAATG
zot PZot-225F TCGCTTAACGATGGCGCGTTTT 946 Rivera et al. (2001)
PZot-1129R AACCCCGTTTCACTTCTACCCA
ompU pOmpU-80F ACGCTGACGGAATCAACCAAAG 868 Rivera et al. (2001)
pOmpU-906R GCGGAAGTTTGGCTTGAAGTAG
ctxA VCT1 ACAGAGTGAGTACTTTGACC 308 Hoshino et al. (1998)
VCT2 ATACCATCCATATATTTGGGAG
O1-rfb O1F2-1 GTTTCACTGAACAGATGGG 192 Hoshino et al. (1998)
O1R2-2 GGTCATCTGTAAGTACAAC
O139-rfb O139F2 AGCCTCTTTATTACGGGTGG 449 Hoshino et al. (1998)
O139R2 GTCAAACCCGATCGTAAAGG
ompW ompW-F CACCAAGAAGGTGACTTTATTGTG 588 Nandi et al. (2000)
ompW-R GAACTTATAACCACCCGCG
ctxA CtxA-F CTCAGACGGGATTTGTTAGGCACG 302 Shirai et al. (1991);
Nandi et al. (2000)
CtxA-R TCTATCTCTGTAGCCCCTATTACG
16S rDNA
a
16S-F CAGCMGCCGCGGTAATWC 888 Amann et al. (1995)
16S-R ACGGGCGGTGTGTRC
a
Currently, there are no published 23S rDNA PCR primers specic for V. cholerae.
the number of presumptive V. cholerae isolates that are not V. cholerae. In this method,
crude template is prepared by boiling to lyse the cells. Genomic regions within this
template are amplied using PCR primers specic to V. cholerae and targeting the
internal transcribed spacer (ITS) region between 16S and 23S rDNA or the outer mem-
brane protein subunit W (ompW). Conrmed V. cholerae strains can be screened for the
genes associated with virulence and their variants (Table 6A.5.3). A more comprehen-
sive list of relevant targets, with PCR conditions and expected amplicons, is provided in
Table 6A.5.4 (http://www.currentprotocols.com/protocol/mc06A05). PCR products are
analyzed by gel electrophoresis and visualized under UV light with ethidium bromide.
Positive and negative controls should be run in parallel and should include eubacterial
16S rDNA PCR reaction for each sample to test template quality (see Table 6A.5.3
for PCR primers, expected amplicon size and reference). Further, phenotypic and ge-
netic screens on V. cholerae isolates can be done following the methods described in
UNIT 6A.3.
6A.5.17
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.
Detection of Vibrio
cholerae from the
Environment
6A.5.20
SUPPORT
PROTOCOL 1
Preparation of Crude DNA Template by Boiling
A crude DNA template, suitable for analysis in Basic Protocol 4, Alternate Protocol 2,
and Basic Protocol 5, can be prepared by boiling a small overnight culture or a loopful
on culture from a fresh agar plate.
Materials
1-ml overnight culture or loopful of pure culture from agar plate (Basic Protocol 2)
2-ml microcentrifuge tubes, sterile
Boiling water bath
1a. From broth: Centrifuge a 1-ml culture (overnight growth at 35
C) and resuspend in
1-ml sterile water.
1b. From agar plates: Resuspend a loopful of pure culture (50 to 100 colonies) of
suspected or presumptive V. cholerae into 300-l of sterile water by vigorously
vortexing.
Plates should be a fresh overnight subculturing demonstrating only one morphology.
2. In a sterile 2-ml microcentrifuge tube, dilute the suspension 1:1000 in sterile water.
Alternatively, a single isolated colony can be resuspended into 20 l of sterile water.
3. Place the microcentrifuge tube containing the resuspended culture into a boiling
water bath for 10 min.
4. Cool tube to room temperature by allowing tube to sit on bench (approximately
30 min).
Treatment of crude DNA template with 10 mg/ml bovine serum albumin (BSA) (4 l
per 100 l supernatant) may help to limit PCR inhibition and yield more accurate
results. It is also benecial to make a 1:10 dilution and a 1:1000 dilution (super-
natant:sterile deionized water) and use this for PCR; dilution may also help to remove PCR
inhibitors.
BASIC
PROTOCOL 4
Vibrio choleraeSpecic PCR ITS
The following PCR methods (Chun et al., 1999) can be used to conrm isolates
as V. cholerae by targeting the internal transcribed spacer (ITS) region between
16S and 23S rDNA or the outer membrane protein subunit W (ompW) specic to
V. cholerae.
Materials
Crude DNA template (Support Protocol 1) or extracted genomic DNA (Basic
Protocol 8)
10 PCR amplication buffer (APPENDIX 2A)
25 mM dNTPs (APPENDIX 2A)
20 M PCR primers (Table 6A.5.3)
Taq DNA polymerase
Molecular weight ladder (e.g., Hyperladder IV, Bioline)
TAE buffer (APPENDIX 2A)
1 g/ml ethidium bromide staining solution (APPENDIX 2A)
Thermal cycler (BioRad)
Microcentrifuge tubes
Boiling water bath
PCR tubes
Nonenteric
Gamma
Proteobacteria
6A.5.21
Horizontal agarose gel apparatus, gel tray and comb (see Voytas, 2000)
Power supply for gel apparatus
UV transilluminator (UVP)
Additional reagents and equipment for agarose gel electrophoresis (Voytas, 2000)
1. Set up V. cholerae-specic ITS (Internal Transcribed Spacer region) PCR reaction
mixture in a total reaction volume of 25 l, containing the following.
5 l DNA template
1 PCR amplication buffer
200 M dNTPs
800 nM primers (pVC-F2, pVCM-R1; Table 6A.5.3)
0.625 U Taq DNA polymerase.
2. Amplify the V. cholerae-specic ITS target with the following cycling conditions:
Initial denaturation: 1 min 94
C (initial denaturation)
30 cycles: 1 min 94
C (denaturation)
1 min 60
C (annealing)
1 min 72
C (extension)
1 cycle: 10 min 72
C (nal extension).
All-in-one PCR master mix can be purchased as an alternative to buying the neces-
sary reagents separately. This can facilitate screening large numbers of strains. PCR
primers and nuclease-free water will need to be purchased separately. Several prod-
ucts include, but are not limited to, GoTaq Green Master Mix (Promega) and Mul-
tiplex PCR 5 Master Mix (New England BioLabs) for detecting multiple targets
by PCR.
Using GoTaq Green Master Mix (Promega), prepare the PCR reaction as follows for a
total reaction volume of 25 l; 5 l template DNA (step 4, 12.5 l 2 GoTaq Master
Mix (containing GoTaq DNA Polymerase, 400 M dATP, 400 M dGTP, 400 M dCTP,
400 M dTTP, 2 mM MgCl
2
), 1l of each primer (20 M pVC-F2 and pVCM-R1), and
5.5 l nuclease free water.
Perform agarose gel electrophoresis and visualize results
3. Run PCR product out on a 1.5% agarose gel in 1 TAE for 1 to 2 hr at 5 V/cm
(Voytas, 2000), along with a molecular weight ladder.
4. Stain the gel in 1 g/ml ethidium bromide staining solution for 15 min.
Alternative DNA-staining dyes that may be less mutagenic have recently been developed
and can be explored as an alternative to ethidiumbromide. Dyes include GelRed and Gel-
Green (Biotium) and SYBR Safe DNA Gel Stain (Invitrogen). Gels stained with GelGreen
can be visualized with a laser-based gel scanner or a Dark Reader that uses visible blue
light for excitation. Gels stained with SYBR Safe DNA Gel Stain can be visualized under
blue light as well.
5. Destain the gel in distilled water for 15 min.
6. Visualize the products by viewing the gel using a handheld UV lamp, transillumina-
tor, or gel documentation system.
The V. cholerae 16S-23S rDNA intergenic spacer region amplicon is 300-bp in size
(Fig. 6A.5.4, lane 2).
7. Screen ITS-PCR conrmed V. cholerae isolates for the virulence-associated factors
listed in Table 6A.5.3 and/or Table 6A.5.4. See Figure 6A.5.4 for image of typical
gel.
Detection of Vibrio
cholerae from the
Environment
6A.5.22
1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10
Figure 6A.5.4 Results of PCR assays used to detect and characterize V. cholerae. Lane 1,
Hyperladder IV (Bioline); lane 2, V. cholerae-specic ITS; lane 3, ctxA (pCTA); lane 4, tcpA of
V. cholerae O1 Classical, lane 5, tcpA of V. cholerae O1 El Tor; lane 6, tcpA of V. cholerae O139;
lane 7, toxR; lane 8, zot; lane 9, ompU; lane 10, O1-O139/ctxA multiplex of V. cholerae O1 and
O139.
ALTERNATE
PROTOCOL 2
Multiplex PCR Assay for Detection of ompW (V. cholerae-Specic) and ctxA
(Toxigenicity)
This protocol (Nandi et al., 2000) can be used in place of Basic Protocol 4, since it detects
the V. cholerae-specic ompW sequence. It also allows for the detection of the ctxA gene
of the cholera toxin.
Materials
Protocol 8)
OmpW-F and R primers
ctxA F and R primers
Taq DNA polymerase
1.5% agarose gel (Voytas, 2000)
TAE buffer (APPENDIX 2A)
1 g/ml ethidium bromide staining solution
Thermal cycler
PCR tubes
UV transilluminator
1. Set up ompW-ctxA multiplex PCR in a total reaction volume of 25 l, containing the
following:
10 to 20 ng of crude DNA template or extracted genomic DNA
Nonenteric
Gamma
Proteobacteria
6A.5.23
250-M dNTPs
1.2-pmol/l of ompW (ompW-F, R) primers
0.25-pmol/l ctxA (ctxA-F, -R) primers
0.625 U Taq DNA polymerase.
2. Amplify the targets with the following cycling conditions:
1 cycle: 5 min 94
30 cycles: 30 sec 94
C (denaturation)
30 sec 64
C (annealing)
30 sec 72
C (extension)
1 cycle: 7 min 72
C (nal extension).
3. Run PCR product out on a 1.5% agarose gel in 1 TAE for 1 to 2 hr at 5-V/cm
(Voytas, 2000) ), along with a molecular weight ladder.
4. Stain the gel 15 min in 1 g/ml ethidium bromide staining solution.
5. Destain the gel 15 min in distilled water.
6. Visualize the products by viewing the gel under UV light.
The ompW and ctxA amplicons are 588 and 302-bp in length, respectively.
Screen samples giving a positive result for isolation of V. cholerae using the traditional
culture method as described in Basic Protocol 2, if desired.
BASIC
PROTOCOL 5
Multiplex PCR Assay for Detection of O1 and O139 Serogroup V. cholerae
and ct xA
The multiplex PCR assay (Hoshino et al., 1998) is performed to conrm O1 and O139
somatic antigens and for the simultaneous detection of the -subunit of the cholera toxin
gene sequence, ctxA in conrmed V. cholerae isolates.
Materials
Protocol 8)
20 M PCR primers (Table 6A.5.3)
Taq DNA polymerase
1 g/ml ethidium bromide solution
Boiling water bath
PCR tubes
Horizontal gel apparatus, gel tray and comb (see Voytas, 2000)
Power supply for gel apparatus
UV transilluminator (UVP)
Detection of Vibrio
cholerae from the
Environment
6A.5.24
1. Set up O1/O139-rfb/ctxA multiplex PCR in a total reaction volume of 30 l, con-
taining the following:
10 to 20 ng of crude DNA template or extracted genomic DNA:
210 M dNTPs
0.5 M O1-rfb (O1F2-1, O1R2-2) primers
0.27 M O139-rfb (O139F2, O139R2) primers
0.17-M ctxA (VCT1, VCT2) primers
0.75 U Taq polymerase.
2. Amplify the targets with the following cycling conditions:
1 cycle: 5 min 94
35 cycles: 1 min 94
C (denaturation)
1 min 55
C (annealing)
1 min 72
C (extension)
1 cycle: 7 min 72
C (nal extension).
3. Run PCR product out on a 2.0% agarose gel in 1 TAE for 1 to 2 hr at 5-V/cm
(Voytas, 2000), along with a molecular weight ladder.
4. Stain the gel 15 min in 1 g/ml ethidium bromide staining solution.
5. Destain the gel 15 min in distilled water.
6. Visualize the products by viewing the gel under UV light.
The O1-rfb, O139-rfb, and ctxAamplicons are 192, 449, and 308-bp in length, respectively
(see Fig. 6A.5.4).
REAL-TIME PCR FOR DETECTION OF V. CHOLERAE
DNA-based methods such as PCR have been increasingly employed. These are de-
signed for rapid, sensitive analysis of a range of clinical and environmental samples.
End-point PCR is not quantitative, and the presence of the PCR product(s) must be
veried using a procedure such as southern hybridization and gel electrophoresis (Lyon,
2001).
It is important to note that, as in the case of conventional PCR, real-time PCR is not
able to discriminate between culturable cells versus VBNC and dead cells, or naked
DNA (that may amplify if the nucleic acid is not completely degraded), which can affect
estimation in a quantitative assay. Furthermore, the presence of substances that inhibit
PCR, often present in environmental samples, can also result in a false negative (Lyon,
2001). Few studies have been designed and validated for a quantitative real-time PCR
method for detection and quantication of V. cholerae. Oligonucleotide primers and
probes for real-time PCR assays are listed in Table 6A.5.5.
NOTE: We highly recommend conducting all real-time PCR tests in a UV hood,
as well as sterilizing all equipment (making sure to not expose reagents, primers,
or probes to ultraviolet radiation) used for real-time PCR. This reduces risk of
contamination.
Nonenteric
Gamma
Proteobacteria
6A.5.25
Table 6A.5.5 Real-Time PCR Primers and Probes
Primer/probe/
beacon name
Sequence Reference
hylA-probe FAM-TCAACCGATGCGATTGCCCAAGA-TAMRA
a
Lyon (2001)
hylA-F-primer TGCGTTAAACACGAAGCGAT
hylA-R-primer AAGTCTTACATTGTGCTTGGGTCA
MBrtxA CGCGATCACCAGAGCGCCAAGAAGTGACTCGTAGATCGCG
b
Gubala and Proll (2006)
MBepsM CGCGATGCCACCGACATCGTAACGCTCCGATCGCG
c
MBompW CCGAAGAAACAACGGCAACCTACAAAGCTTCGG
d
MBtcpA CGCGACGCTGAAACCTTACCAAGGCTGACCAAGTCGCG
e
rtxA V1F AGCAAGAGCATTGTTGTTCCTACC
rtxA V1R ACTTCCCTGTACCGCACTTAGAC
epsM V3F TGGTTGATCGCTTGGCGCATC
epsM V3R ATGGCAGCCTTTGAGTGAG
mshA V6F ACACCTGGAACAGTTATTGATGGC
mshA V6R TCACTCGAAGTATCTAGCGTTTGC
tcpA V7F TGCAATGACACAAACTTATCGTAG
tcpA V7R CCCATAGCTGTACCAGTGAAAG
a
The TaqMan V. cholerae-specic probe is an oligonucleotide with a 5
reporter dye (FAM-6-carbooxyuorescein) and a 3
quencher dye
(TAMRA-6-carboxy-N
-tetramethylrhodamine).
b
FAM (6-carboxyuorescein) uorophore and Dabcyl quencher.
c
Texas Red uorophore and BHQ2 quencher.
d
Cy5 uorophore and BHQ3 quencher.
e
Cy3 uorophore and BHQ2 quencher.
BASIC
PROTOCOL 6
TaqMan Assay for Detection of V. chol erae
The TaqMan assay (Lyon, 2001) is a real-time PCR method based on 5
exonuclease
activity of Thermus aquaticus DNA polymerase used to hydrolyze an internal probe
labeled with a uorescent reporter dye (FAM, Cy3, Cy5, TET) and a quencher dye. During
PCR amplication, hydrolyis of the probe separates the reporter dye from the quencher
dye, which results in increasing uorescence proportional to the amount of template DNA
present in the reaction, thereby giving a quantitative estimation. Lyon (2001) developed
a method for a quantitative, sensitive, and rapid detection of V. cholerae in pure cultures,
seawater, and raw oysters, with primers and probes directed toward the non-classical
specic hemolysin (hylA) gene of V. cholerae O1, O139, and non-O1/O139 (Table
6A.5.5). Another quantitative TaqMan assay was developed for detection of the ctxA
gene in seawater and oysters with the sensitivity of <10 CFU per reaction (Blackstone
et al., 2007).
Materials
100 ng/l extracted DNA sample (Basic Protocol 8)
TaqMan PCR Reagent Kit (Applied Biosystems) containing:
10 TaqMan buffer A
25 mM MgCl
2
dATP, dCTP, dGTP, and dUTPs
AmpliTaq Gold DNA polymerase
1 U/l AmpErase uracil N-glycosylase (UNG)
hylA forward primer (Table 6A.5.5)
Detection of Vibrio
cholerae from the
Environment
6A.5.26
hylA reverse primer (Table 6A.5.5)
hylA probe (FAM; Table 6A.5.5)
Optical reaction tubes or plates
Optical reaction tube caps or plate seal
Real-time PCR (qPCR) machine
1. Set up real-time PCR with a total amplication reaction mixture of 50-l per sample
containing the following:
2.5 l 100 ng/l DNA sample
1 TaqMan buffer A
5 mM MgCl
2
200 M (each) dATP, dCTP, and dGTP
400 M dUTP
0.02 M hylA-probe
0.3 M hylA-F primer
0.3 M hylA-R primer
1 U of AmpErase uracil N-glycosylase
2.5 U of AmpliTaq Gold DNA polymerase.
2. Amplify the targets with the following cycle conditions:
1 cycle: 5 min 50
C (initial hold)
1 cycle: 5 min 94
C (denaturation)
1 min 60
C (annealing).
1
D
e
l
t
a

R
n
Cycle number
Delta Rn vs. cycle
123456789
1
0
1
1
1
2
1
3
1
4
1
5
1
6
1
7
1
8
1
9
2
0
2
1
2
2
2
3
2
4
2
5
2
6
2
7
2
8
2
9
3
0
3
1
3
2
3
3
3
4
3
5
3
6
3
7
3
8
3
9
4
0
4
1
4
2
4
3
4
4
4
5
4
6
4
7
4
8
4
9
5
0
0
1
2
3
4
5
6
Figure 6A.5.5 Real-Time PCR amplication curves of V. cholerae spiked in lter-sterilized water.
Nonenteric
Gamma
Proteobacteria
6A.5.27
3. Interpret results.
Results are interpreted using software available with real-time PCR machines by monitor-
ing increase in uorescence throughout the amplication cycles and reporting C
t
value.
The C
t
value is the number of amplication cycles required to detect a uorescent signal
above a given threshold. C
t
levels are inversely proportional to the amount of target
nucleic acid in the sample. In general, C
t
values <29 are considered strong positive re-
actions and are indicative of abundant target nucleic acid in the sample, while C
t
values
of 30 to 35 are positive reactions indicative of moderate amounts of the target, and C
t
values of 38 to 40 are considered weak reactions with little or no target nucleic acid in
the sample (Fig. 6A.5.5).
BASIC
PROTOCOL 7
SYBR Green Assay for Detection of V. chol erae
An alternative real-time PCR method (Gubala, 2006) involves the use of a dsDNA-
binding dye such as SYBR Green I. In this technique the amplication products are
distinguishable by analysis of their melting temperature. This assay was used in two
studies to develop a multiplex real-time PCR of four and six target genes for V. cholerae
and other vibrios detected in sea, estuarine, and river water samples, and in seafood
(Table 6A.5.5; Gubala, 2006; Gubala and Proll, 2006).
Materials
Extracted DNA sample (Basic Protocol 8; for optimal results, a serial dilution of
various concentrations of DNA samples is suggested)
LightCycler FastStart DNA Master SYBR Green I (Roche Diagnostics) including:
LightCycler FastStart Enzyme
LightCycler FastStart Reaction Mix SYBR Green
MgCl
2
Stock Solution, 25 mM
PCR-grade Taq DNA polymerase
rtxA forward and reverse primer (Table 6A.5.5)
epsM forward and reverse primer (Table 6A.5.5)
mshA forward and reverse primer (Table 6A.5.5)
tcpA forward and reverse primer (Table 6A.5.5)
Optical reaction tubes or plates
Optical reaction tube caps or plate seal
Smart Cycler (Cepheid)
1a. For a single target: Prepare 25-l amplication reaction mixtures containing:
1 l of template DNA
0.2 M of each primer
2.5 l of LightCycler FastStart DNA Master SYBR Green I
3.75 mM MgCl
2
(nal concentration).
1b. For multiplex PCR: Prepare 25-l amplication reaction mixtures containing:
1 l template DNA.
0.08 M each rtxA primer
0.15 M each epsM primer
0.40 M each mshA primer
0.20 M each tcpA primer
3 l of LightCycler FastStart DNA Master SYBR Green I
4.0 mM MgCl
2
(nal concentration).
Detection of Vibrio
cholerae from the
Environment
6A.5.28
2a. For a single target: Amplify the target with the following cycling conditions:
1 cycle: 150 sec 95
C (denaturation)
30 sec 60
C (annealing)
30 sec 72
C (extension).
2b. For multiplex assay: Amplify the targets with the following cycling conditions:
1 cycle: 150 sec 95
C (denaturation)
30 sec 60
C (annealing)
30 sec 72
C (extension).
3. Interpret results.
Results are interpreted using designated computer software. As with the TaqMan assay,
results are expressed in C
t
values, the number of amplication cycles required to detect a
uorescent signal above a given threshold. In a multiplex SYBR Green real-time PCR, the
amplication products are distinguished based on their melting temperature (T
m
). The
T
m
values of each of the products are calculated at the completion of PCR amplication,
monitoring the uorescence in increasing temperature between 60
Cand 95
C, at a given
rate. The T
m
peaks are calculated based on the initial uorescence curve.
BASIC
PROTOCOL 8
DIRECT PCR FOR ENVIRONMENTAL SAMPLES
Three types of targets are used to detect V. cholerae in environmental samples by PCR:
species-specic genes (16S rDNA, ITS, ompW); serogroup-specic genes (O1 and O139
wbe); and toxin and pathogenic factor genes (ctx, tcpA, etc.). Briey, water, plankton,
oyster, and/or sediment samples are collected and concentrated. DNA is extracted from
the samples, using a modication of the method of Murray and Thompson (1980), and
PCR is performed on the extracted DNA using a multiplex (ompW and ctxA) primer
array. The PCR protocol described in Basic Protocol 5 is used, and the DNA template is
derived from the DNA extraction method described below. Positive and negative controls
should be run in parallel, and should include a eubacterial 16S rDNA PCR reaction on
each sample to test template quality. See Table 6A.5.3 for PCR primers and references.
Materials
Water, plankton, sediment, or oyster samples
TE buffer (APPENDIX 2A)
10% (w/v) SDS
20 mg/ml (2% w/v) proteinase K solution
5 M NaCl
CTAB/NaCl solution (see recipe)
25:24:1 phenol/chloroform/isoamyl alcohol (APPENDIX 2A)
24:1 chloroform/isoamyl alcohol
Isopropanol
70% ethanol
65
C water bath or heat block

Boiling water bath
Vacuum desiccator or lyophilizer
Additional reagents and equipment for sample collection (Basic Protocol 1),
bacteriological culture method for V. cholerae (Basic Protocol 2), and PCR
assays (Basic Protocol 4 or 5 or Alternate Protocol 2)
Nonenteric
Gamma
Proteobacteria
6A.5.29
Collection and concentration of environmental sample
For water and plankton samples
1a. Collect sample as described in the conventional bacterial culture method above
(Basic Protocol 1).
2a. Perform enrichment in APW using the conventional bacterial culture method (Basic
Protocol 1).
For sediment samples
1b. Add sediment to 100-ml of distilled water until the nal volume reaches 200 ml. Mix
well and allow the sediment to settle. Remove particulate matter by centrifuging a
10-ml aliquot of the slurry for 8 min at 1000 g, room temperature.
2b. For sediment samples, enrich 1 ml of the sediment slurry in an APW enrichment
ask, as for water and plankton samples (Basic Protocol 1).
For oyster samples
1c. Collect sample as described in the conventional bacterial culture method above
(Basic Protocol 1)
2c. Perform enrichment in APW using the conventional bacterial culture method (Basic
Protocol 1).
Processing these samples in 10-fold dilutions and in triplicate or quintuplicate (i.e.,
triplicates or quintuplicates of 100, 10, and 1 ml of water), followed by direct PCR
on these dilutions, will allow a quantitative estimation of the Most Probable Number
(MPN) PCR target in each sample. This can be done for all collected sample types, and
dilutions for oysters and sediment would be 10-fold weight increments (i.e., 10, 1, 0.1 g
of homogenized oysters). For more information on the Most Probable Number assay see
Standard Methods for the Examination of Water and Wastewater (Clesceri et al., 1998).
It is possible to perform a quick DNA extraction at this point, and proceed directly to PCR
(step 14), skipping DNA purication:
1. Remove a 1-ml aliquot from the upper surface (i.e., top 1 to 2 mm) of the APW
enrichment.
2. Boil for 10 min.
3. Put on ice, then centrifuge to pellet cell material.
4. Remove 700 l supernatant and add this to a sterile 1.5-ml microcentrifuge tube.
This supernatant contains community DNA.
5. Add 28 l of 10 mg/ml bovine serum albumin (BSA) (4 l per 100-l supernatant).
It is also benecial to make a 1:10 dilution (supernatant:sterile DI water) and use this
for PCR as well, as dilution may help to reduce the concentration of PCR inhibitors that
can be present in community DNA samples.
DNA extraction using phenol, chloroform, and isoamyl alcohol
3. Microcentrifuge a 1-ml aliquot from the upper surface (i.e., top 1 tob 2 mm) of the
APW enrichment for 5 min at 12,000 g, room temperature.
4. Resuspend the cell pellet in 567 l TE buffer.
5. Add 30 l of 10% SDS, followed by 3 l of 20 mg/ml proteinase K solution.
6. Incubate the suspension 1 hr at 35
C.
7. Add 100 l of 5 M NaCl, followed by 80 l of CTAB/NaCl solution.
8. Incubate mixture 10 min at 65
C.
Detection of Vibrio
cholerae from the
Environment
6A.5.30
9. Add 800 l phenol/chloroform/isoamyl alcohol (25:24:1), vortex, and microcen-
trifuge 5 min at 12,000 g, room temperature.
10. Transfer aqueous phase (supernatant) to a new microcentrifuge tube. Add 800 l
of chloroform/isoamyl alcohol (24:1), vortex, and microcentrifuge 5 min at 12,000
g, room temperature.
11. Transfer aqueous phase (supernatant) to a new microcentrifuge tube. Precipitate
DNA with an equal volume of isopropanol.
12. Pellet DNA by centrifuging 5 min at 12,000 g, 25
C. Wash DNA with 1 ml of

70% ethanol and microcentrifuge 5 min at 12,000 g, 25
C.
13. Dry pellet in vacuum desiccator or lyophilizer and resuspend in 100 l TE buffer.
Perform direct PCR
14. Perform the PCR protocols described in Basic Protocol 4 or Alternate Protocol 2 or
Basic Protocol 5 on the extracted DNA template.
Additionally, PCR assays described in Table 6A.5.4 can be explored to evaluate the
presence of other V. cholerae virulence genes not targeted in these three protocols.
BASIC
PROTOCOL 9
COLONY BLOT HYBRIDIZATION WITH LABELED RNA OR DNA PROBES
The colony lift procedure is used to immobilize DNA from bacterial colonies onto ni-
trocellulose or nylon lters to allow quick screening of a large number of colonies for
genetic elements of interest by hybridization. The colony blot hybridization procedure
is culture based, so it is dependent upon the presence of V. cholerae in the sample as vi-
able, culturable cells. Its detection by hybridization precludes the necessity of numerous
biochemical tests. Its advantage over PCR is that isolation is performed simultaneously
with blot preparation, and enumeration can be performed more easily. Briey, LB or
modied nutrient agar spread plates are prepared from water samples and incubated
overnight. Other plating media can be used, but the medium should be relatively rich
and nonselective to allow for vigorous growth and cells with a high RNA content. Nitro-
cellulose (or nylon) membranes are overlaid, lifted, and treated to bind RNA (Rehnstam
et al., 1989) (or DNA) to the membrane. Plates to be lifted should contain 50 to 150
well-dened colonies, 2.0- to 3.0-mm in size. Membranes should be handled with sterile
forceps only, and can be sterilized in an autoclave between two pieces of lter paper
for 15 min prior to use. For RNA blots, care should be taken to minimize RNase con-
tamination. Blots are then hybridized with labeled probe specic for V. cholerae (and V.
mimicus), 5
-ACTTTGTGAGATTCGCTCCACCTCG-3
(Heidelberg, 1997; Heidelberg

et al., 2002), or toxigenic V. cholerae (ctxA). V. mimicus is a closely related species to
V. cholerae, which was previously described as biochemically atypical V. cholerae (non-
sucrose-fermenting, indicated by green colonies instead of yellow colonies on TCBS). V.
mimicus produces a variety of toxins, including cholera toxin (potential reservoir), and
has caused sporadic diarrhea (Ramamurthy et al., 1994). Fluorescently labeled probes are
preferred (e.g., see Boyle and Perry-OKeefe, 1992); however, the DIG system (Roche)
offers a good alternative when a variable mode imager (such as Typhoon, GE Health-
care) or an alternative machine, such as Dark Reader (Clare Chemical Research), for
detection of the uorochrome, is not available. The V. cholerae-specic RNA colony
blot/hybridization protocol is presented rst, and then a ctxA-DNA colony blot/DIG
(Roche) hybridization protocol is presented.
Materials
Bacteria growing in APW enrichment asks (Basic Protocol 2) or water sample for
enumeration
Nonenteric
Gamma
Proteobacteria
6A.5.31
LB or modied nutrient agar plates (see APPENDIX 2C)
10% (w/v) SDS
3 SSC (APPENDIX 2A)
Pre-washing solution (see recipe)
DEPC-treated water (APPENDIX 2A)
Hybridization solution base (see recipe)
Washing solution (see recipe)
Fluorescein-labeled Vchomim1276 probe, reconstituted at 25 ng/l (see recipe)
85-mm sterile nitrocellulose membranes, 0.22-m (GE Osmonics)
65
C incubator
Filter paper (Whatman)
Pyrex dish slightly larger than membrane
70
C oven
Typhoon scanner (GE Healthcare) or Dark Reader (Clare Chemical)
NOTE: All solutions should be DNase and RNase-free. For RNA colony blot hybridiza-
tion, use DEPC-treated water (see APPENDIX 2A) to make hybridization solutions.
Spread-plate preparation
1. For detection, prepare spread plates from APW enrichment asks using APW as
diluent and plating three serial-fold dilutions onto LBor modied nutrient agar plates.
2. For enumeration (and detection), spread plate appropriate dilutions of water sample
onto LB or modied nutrient agar plates on-site without APW enrichment.
Alternatively, 100 to 500 ml of water may be ltered through 0.22-m nylon membranes
and overlaid onto an agar plate. If this method is preferred, incubate membrane and plate
overnight at 30
C and then proceed to step 7.

3. Incubate plates overnight at 30
C.
Colony lift
4. Mark membranes using a lead pencil with a Blot ID (e.g., medium, sample, dilution)
that matches plate to be lifted and orientation marks (asymmetrical).
5. Overlay nitrocellulose membrane, starting from the center, to ensure there are no air
bubbles.
6. Allow at least 15 min for transfer.
7. Replica plate the membrane onto a fresh modied nutrient agar plate, transferring
orientation markings.
8. Preheat SDS, SSC, and Pyrex dishes (for holding lter paper) to 65
C.
9. Place membrane, colony-side-up, on the lter paper (cut to just slightly larger than
the membrane) pre-wetted with 10% SDS, enclosed in a Pyrex dish, for 5 min at
65
C. Cover Pyrex dish containing wetted lter paper and membrane with Saran
wrap or equivalent plastic lm to prevent the lter paper from drying out.
It is important to use lter paper pre-wetted, but not saturated, with 10% SDS or 3SSC
(see next step) to prevent colonies from over-swelling and losing their circular shape.
Pour or pipet the liquid onto the lter paper, let soak briey, remove air bubbles, and then
pour off excess. Ensure that there is no pooled liquid on the lter paper prior to placing
membrane.
10. Incubate the membrane 15 min at 65
C on fresh lter paper wetted with 3 SSC.

Cover Pyrex dish containing wetted lter paper and membrane with Saran wrap or
equivalent plastic lm to prevent the lter paper from drying out.
Detection of Vibrio
cholerae from the
Environment
6A.5.32
11. Let membranes air dry 10 min on lter paper.
12. Bake membranes 15 min at 70
C.
RNA-colony blot hybridization
13. Wash membranes three times in adequate pre-washing solution for 15 min at room
temperature.
14. Wash membranes 1.5 hr at 60
C in pre-washing solution.
15. After pre-washing steps, rinse membranes in DEPC-treated water.
Hybridization solution base will form a precipitation upon maintaining at room temper-
ature. If this happens, heat to 40
to 50
C to resuspend.
16. Pre-hybridize the membranes 30 min at 60
C in hybridization solution base at a ratio

of 10-ml solution per 100-cm
2
of blot membrane.
85 mm-membranes have a surface area of 60-cm
2
.
17. Pour off solution and add hybridization solution base plus uorescein-labeled
Vchomim1276 probe (32 l of 25 ng/l probe per 10 ml solution) at a ratio of
10 ml solution per 100-cm
2
blot.
18. Hybridize overnight (ideally 16 to 20 hr) at 60
C.
19. After hybridization, wash membranes 30 min at 60
C in washing solution.
20. View/image the membrane using a Typhoon Scanner or Dark Reader.
21. From replica plates, subculture colonies that were positive by blot hybridization for
further analysis, if desired.
ALTERNATE
PROTOCOL 3
COLONY BLOT HYBRIDIZATION USING DIG-LABELED ct x DNA
PROBE
The previous colony blot hybridization protocol (Basic Protocol 9) is used to detect
V. cholerae and closely related V. mimicus. This protocol, on the other hand, targets only
toxigenic strains of V. cholerae. The presence of ctxA is conrmed by hybridization using
a ctxA-specic DNA-probe. There may be some cross-reactivity of the probe with the
heat-labile toxin (LT) of E. coli (Dallas and Falkow, 1980). Colony blots are prepared
following the method of Pal et al. (1992). The hybridization is done according to the
DIG protocol (Roche). The protocol is given here; readers are encouraged to consult
the manual accompanying the High Prime kit. The DIG DNA probe-labeling protocol is
given in Support Protocol 2. The ctxA probe can be produced by PCR, using the pCTA
primer set (see Table 6A.5.3) or by EcoRI digestion of the plasmid, pKTN901, which
contains a 540-bp XbaI-ClaI fragment of ctxA (Kaper et al., 1988).
Materials
Bacteria growing in APW enrichment asks (Basic Protocol 2)
Luria-Bertani (LB) agar plates (APPENDIX 4A)
Cell lysis buffer (see recipe)
Blot neutralization solution (see recipe)
1 SSC buffer (APPENDIX 2A)
Proteinase K solution (see recipe)
DIG High Prime DNA Labeling and Detection Starter Kit II (Roche) kit includes:
DIG Easy Hyb granules
10 blocking solution
CSPD
Nonenteric
Gamma
Proteobacteria
6A.5.33
Anti-digoxigenin-AP conjugate
DIG High Prime
Labeled ctxA probe (Support Protocol 2)
Stringency wash solution I (see recipe)
Stringency wash solution II (see recipe)
Maleic acid buffer (see recipe)
Antibody solution: 1:10,000 anti-digoxigenin-AP conjugate in 1 blocking
solution (from kit)
Washing buffer (see recipe)
Detection buffer (see recipe)
Incubators, 37
C and 42
C
Sterile nylon (or nitrocellulose) membranes, 85mm, 0.22-m (GE Osmonics)
Whatman lter paper, #3
UV cross-linker or transilluminator
Shaking water bath
Hybridization pouch
X-ray lm (Kodak or Fuji)
Film developer
Blot preparation
1. Prepare spread plates as above, using LB agar plates (see Basic Protocol 9, steps 1
to 3).
2. Incubate the plates overnight at 37
C
3. Mark membranes using a lead pencil, including Blot ID (e.g., medium, sample,
dilution) that matches plate to be lifted and orientation marks (asymmetrical).
4. Overlay membrane, starting from the center to ensure there are no air bubbles.
5. Transfer at least 15 min.
6. Transfer the blot onto a new LB plate, keeping colony side up, and incubate 3 hr at
37
C. Wrap the master plate with Paralm and keep at 10
to 15
C.
Master plates can be kept like this for up to 2 weeks.
7. Place membranes, colony side up, onto #3 Whatman lter paper pre-soaked with cell
lysis buffer. Incubate 10 min at room temperature.
8. Remove the membrane from cell lysis buffer and place onto #3 Whatman lter paper
pre-soaked with blot neutralization solution
9. Repeat step 8.
10. Remove membrane from blot neutralization solution, place onto #3 Whatman lter
paper, and air dry (30 min).
11. Immobilize colonies onto the membrane using a UV cross-linker or transilluminator.
Damp membranes should be cross-linked at an output intensity of 120-mJ/cm
2
, which
corresponds to the optimal or auto-crosslink setting on commercially calibrated ma-
chines. Transilluminators or hand-held UV lamps can be used, if calibrated; however, the
following times should be sufcient: 1 min for 254-nm lamps or 3 min for 302-nm lamps.
12. Rinse the blot two times in 1 SSC buffer and air dry (30 min).
13. Treat membranes 30 min at 42
C with 100 ml proteinase K solution using gentle

shaking.
Detection of Vibrio
cholerae from the
Environment
6A.5.34
14. Rinse lters three times in 1 SSC in a shaking water bath at room temperature for
10 min each. Let air dry (30 min).
Prehybridization and hybridization
15. Reconstitute DIG Easy Hyb granules according to the manufacturers instructions to
prepare DIG Easy Hyb buffer. Preheat DIG-Easy Hyb buffer to 42
C.
16. Prehybridize blots in preheated DIG-Easy Hyb buffer for 30 min at 42
C with gentle
agitation.
17. Denature the DIG-labeled probe by boiling for 5 min and rapidly cooling on ice.
18. Pour off DIG-Easy Hyb buffer from the blots.
19. Add fresh DIG-Easy Hyb buffer plus denatured, labeled ctxA probe (25 ng/ml solu-
tion) and incubate overnight at 42
C.
Stringency washes
20. Pour off probe-containing hybridization solution and store up to 2 months at 20
C.
Probe-containing hybridization solution can be reused several times.
21. Wash membrane two times in Stringency Wash Solution I for 5 min at room temper-
ature under constant agitation.
22. Wash two times in Stringency Wash Solution II for 15 min at 65
C under constant
agitation.
Detection
23. Rinse membrane briey (5 min) in washing buffer.
24. Prepare 1 blocking solution by diluting 10 blocking solution from the kit in
maleic acid buffer. Incubate the membrane 30 min at 25
C in 1 blocking solution.
25. Incubate the membrane 30 min at 25
C in antibody solution (1:10,000 dilution of

anti-digoxigenin-AP conjugate) for 30 min.
26. Wash the membrane two times, each time for 15 min, in washing buffer, for 15 min
each.
27. Equilibrate the membrane in detection buffer for 5 min at 25
C.
28. Place membrane in hybridization pouch. Add CSPD to the membrane, cover, and
incubate at room temperature for 5 min.
29. Remove excess liquid from pouch, seal, and incubate at 37
C for 10 min.
30. Expose the membrane to X-ray lm for 20 min at room temperature and develop.
If lm is under- or over-exposed, repeat step 30 and vary time of exposure accordingly.
31. From master plates (step 6), subculture colonies that were positive by blot hybridiza-
tion for further analysis.
SUPPORT
PROTOCOL 2
DIGOXIGENIN LABELING OF ct xA PROBE USING DIG-HIGH PRIME
PCR-or digestion-generated probes can be labeled by DIG-High Prime (Roche), ran-
domly incorporating digoxigenin-11-dUTP. A 563-bp ctxA fragment can be produced by
amplifying extracted DNAfroma toxigenic laboratory reference strain. The PCRproduct
should be puried by using a PCR clean-up kit or by gel electrophoresis and extraction.
Alternately, a PCR product may be labeled during the PCR amplication using the PCR
DIG Probe Synthesis Kit (Roche). A 554-bp ctxA probe is also available on a plasmid,
Nonenteric
Gamma
Proteobacteria
6A.5.35
pKTN901(Kaper et al., 1988), which has been widely used (Pal et al., 1992; Islam et al.,
2005). The XbaI-ClaI fragment can be removed from the plasmid by digestion with
EcoRI and gel puried. Labeled probes can then be used in the DIG-based hybridization
above.
Materials
DNA extracted from toxigenic (ctxA+) V. cholerae reference strain (ATCC)
Forward and reverse pCTA primers for amplifying ctxA (Table 6A.5.5)
Plasmid pKTN901 (available from Dr. James Kaper, jkaper@umaryland.edu) or
other source of ctxA probe
EcoRI restriction endonuclease (or other restriction enzyme depending on source
of ctxA probe) and restriction buffer DIG-High Prime (Roche)
0.2 M EDTA, pH 8.0
Boiling water bath
65
C incubator
Additional reagents and equipment for PCR (Kramer and Coen, 2001), agarose gel
electrophoresis (Voytas, 2000), and isolation and purication of fragments from
agarose gels (Moore et al., 2002)
1. For PCR-generated ctxA target fragment, amplify extracted DNA of a toxigenic
(ctxA
+
) V. cholerae reference strain using the pCTA primer set and the master mix
and cycling conditions described in Basic Protocol 4.
2. For digestion-generated ctxA target fragment, digest plasmid, pKTN901, with EcoRI
for 2 hr at 37
C.
3. Clean-up PCR reaction product or digestion product by agarose gel electrophoresis
(Voytas, 2000) followed by gel extraction (Moore et al., 2002).
4. Bring 1-g of puried ctxA fragment up to a total volume of 16-l in sterile distilled
water.
5. Denature by placing in a boiling water bath for 10 min, followed by rapidly chilling
in an ice bath (5 min).
6. Add 4-l of DIG-High Prime to the DNA solution, mix, and centrifuge briey to
collect liquid.
7. Incubate overnight at 37
C.
8. Stop reaction by adding 2 l of 0.2 M EDTA, pH 8.0, and/or by heating to 65
C for
10 min.
IMMUNOLOGICAL METHODS FOR DIRECT DETECTION OF
V. CHOLERAE IN ENVIRONMENTAL SAMPLES
Conventional culture methods are ineffective when bacterial cells have entered into the
viable but non-culturable (VBNC) state. Thus, direct detection becomes extremely im-
portant. Despite the ubiquitous nature of V. cholerae, isolation and detection by traditional
methods are difcult since these methods rely on culturing the organism. The discovery
of monoclonal antibody in the 1980s and, subsequently, the development of a monoclonal
antibody against V. cholerae O1, triggered development of direct detection methods for
this bacterial species (Xu et al., 1984; Hasan et al., 1992). Using immunological meth-
ods, the mystery concerning the inability to culture V. cholerae in environmental samples
was reported during an inter-epidemic period in Bangladesh (Roszak and Colwell, 1987;
Huq et al., 1990). These difculties arise from several possible factors: low density,
inter-specic competition, cell state, and health (VBNC, starved). Fluorescent In-Situ
Detection of Vibrio
cholerae from the
Environment
6A.5.36
Hybridization (FISH) allows direct detection of taxa-specic nucleic acid followed by
visualization using microscopy, and the polymerase chain reaction offers a molecular-
based alternative to the traditional culture and immunological methods (discussed later).
BASIC
PROTOCOL 10
Fluorescent In Situ Hybridization (FISH) Detection of V. chol erae
Direct quantication methods without the need to enrich and culture can yield highly
accurate and quick estimates of taxon-specic densities in water bodies. Fluorescence in
situ hybridization (FISH) accomplishes this with a uorescently labeled oligonucleotide
probe that is visualized under epiuorescence or confocal laser scanning microscopy.
This method is reliable for accurate estimations of V. cholerae cells in media, as it allows
one to quantify both culturable and non-culturable (VBNC) V. cholerae cells.
Materials
Water for testing
4% formaldehyde (see recipe)
PBS-ethanol solution (1:1 PBS:absolute ethanol)
0.01% (w/v) poly-L-lysine (PLL)
Ethanol solutions (50%, 80%, and 96%)
Hybridization solution (see recipe)
FITC-labeled probe: Vchomim1276, 6-FAM (uorescein phosphoramidite) labeled
probe (5
-[5FITC] ACTTTGTGAGATTCGCTCCACCTCG-3
) at a working
concentration of 50 ng/l in sterile water (nal concentration, 5 ng/l)
Washing buffer solution (see recipe)
Citiuor AF1 antifade agent (Electron Microscopy Sciences)
Multiwell slides
Incubator or water bath set to 45
C
Humid chamber: 50-ml centrifuge tube containing one to two strips of
hybridization solutionsaturated lter paper
Whatman lter paper
Sample preparation
1. Filter water (500 to 1000 ml) through a 0.2 m polycarbonate membrane, and
resuspend cells attached to membrane in 5 ml PBS.
2. Centrifuge 1 ml of the PBS suspension 10 min at 13,000 g, room temperature.
Discard supernatant and resuspend in 250 l of PBS.
3. Fix cells by adding 750 l of fresh 4% paraformaldehyde solution and incubate at
room temperature (22
25
C) for 1 hr.
4. Next, pellet the cell solution by centrifugation for 5 min at 13,000 g, room
temperature. Discard the supernatant and then wash the cell twice with PBS using
the same centrifugation conditions.
5. Concentrate the cells by centrifugation and resuspend in 100 l PBS-ethanol solution.
6. Store cells at 20
C.
Hybridization
7. Immerse multiwell slides in 0.01% PLL for 10 min and air dry.
8. Spot 5-l aliquots of xed cells onto slides and air dry.
9. Wash slides for 3 min each in successively higher concentration ethanol solutions
(50%, 80%, and 96%), and then air dry. Prewarm humid chamber, slides, and lter
paper soaked with hybridization solution at 45
C for 10 min.
Nonenteric
Gamma
Proteobacteria
6A.5.37
Figure 6A.5.6 Fluorescent in situ hybridization (FISH) image of V. cholerae cells under epiuo-
rescence microscopy.
10. To each well, add 5 l aliquot of hybridization solution containing 5 ng/l of labeled
probe.
11. Perform hybridization at 45
C for 24 hr in the humid chamber using hybridization

solutionsoaked lter paper.
12. Pre-warm the washing buffer solution at 45
C for 10 min before washing the slides

and incubate at 45
C for 10 min.
13. Wash slides in sterile deionized water and air dry at room temperature.
14. Add antifade agent to each well and apply a coverslip.
15. Visualize uorescence under epiuorescence microscopy (Fig. 6A.5.6.)
BASIC
PROTOCOL 11
Direct Fluorescent AntibodyDirect Viable Count (DFA-DVC) Method
The direct uorescent antibody staining method for rapid detection of V. cholerae
serogroup O1 and O139 is a very useful, direct, and culture-independent method. Coupled
with the direct viable count method of Kogure et al. (1979), it can distinguish culturable,
viable cells from viable but non-culturable cells (VBNC) of V. cholerae (Chowdhury
et al., 1994). It is a two-step method where samples are incubated with yeast extract in
the presence of nalidixic acid, after which actively viable, substrate-responsive cells be-
come enlarged and elongated (Kogure et al., 1979). Next, an aliquot from this suspension
is air dried on a glass slide and stained with uorescently labeled monoclonal antibody,
raised against A factor of V. cholerae O1 lipopolysaccharide, which reacts with both
serotypes, Ogawa and Inaba (Colwell et al., 1990; Hasan et al., 1994). Antibodies against
V. cholerae O139 are also available (Hasan et al., 1995). When observed under an epi-
uorescent microscope, elongated cells of V. cholerae O1 or O139 (based on the type of
antibody used) exhibit a bright-green uorescing periphery (the outer cell wall) with a
dark interior (Fig. 6A.5.7). V. cholerae O1 and O139 DVC-DFA positive samples can be
conrmed by PCR (Binsztein et al., 2004). DFA-DVC is a rapid method by which one
can determine the presence of V. cholerae within 8 hr (when the DVC incubation is 6 hr);
however, overnight incubation with yeast extract and nalidixic acid is preferred. Kits
for V. cholerae O1 (CholeraDFA) and O139 (BengalDFA) DFA tests are commercially
available (New Horizon Diagnostics).
Detection of Vibrio
cholerae from the
Environment
6A.5.38
Materials
Concentrated water or homogenized plankton sample (Basic Protocol 2)
Yeast extract, 2.5% solution in distilled water
Nalidixic acid, 0.2% solution in distilled water
37% to 40% (v/v) formaldehyde solution or fresh 4% formaldehyde (see recipe)
100% methanol
CholeraDFA and/or BengalDFA kit (test kit comes with FITC-conjugated DFA
reagent, positive and negative control, slides and uorescent mounting medium;
New Horizon Diagnostics, http://www.nhdiag.com/)
35
C incubator
Multiwell slides and cover slips
Humid chamber: 50-ml centrifuge tube containing one to two strips of lter paper
saturated with deionized H
2
O
Epiuorescent microscope, with FITC lter set
NOTE: All solutions should be 0.1 m ltered and sterile, as VBNC cells of V. cholerae
appear as small coccoid cells in a size range of 0.1 to 0.8 m.
A B
C D
Figure 6A.5.7 DFA staining of V. cholerae O1 using the CholeraDFA Kit (New Horizon Diagnostics). (A) Fresh culture;
(B) VBNC cells; (C) and (D), DVC-incubated cells.
Nonenteric
Gamma
Proteobacteria
6A.5.39
Perform DVC incubation
1. To 1 ml concentrated water or homogenized plankton sample, add 10 l yeast extract
solution and 10 l nalidixic acid solution.
2. Freeze (20
C) a parallel sample (1 ml) for PCR conrmation.

3. Incubate the mixture at 25
C for a minimum of 6 hr to overnight.

4. Fix the sample by adding formaldehyde to a nal concentration of 3% (v/v) and
incubating 30 min at room temperature in the dark.
Fixed samples can be stored at 4
C in the dark for up to 6 months.

Perform DFA protocol
5. Place 5 to 10 l of the xed sample onto a glass slide and air dry (15 to 20 min).
6. Fix by adding 5 l methanol and air dry (1 to 5 min).
7. Add 10 l of reconstituted FITC-conjugated specic DFA reagent (Hasan et al.,
1994), as supplied by the Cholera DFA or BengalDFA kit.
8. Incubate 30 min at 37
C in humid chamber. Protect slide from light.

9. Rinse slide with 50 ml PBS (each). Air dry slides in the dark (15 to 20 min).
10. Mount slide with one drop of kit-provided uorescent mounting medium and add
coverslip.
11. Observe under an epiuorescent microscope (see Fig. 6A.5.7).
BASIC
PROTOCOL 12
Indirect Fluorescent Antibody (IFA) Method
This immunouorescent method for detection of V. cholerae serogroup O1 in aquatic
environmental samples was rst introduced by Xu et al. (1984). Antiserumspecic for O1
somatic antigen produced in rabbits was used with uorescein-isothiocyanate (FITC)-
conjugated anti-rabbit globulin goat serum, with rhodamine isothiocyanate (RITC)
conjugated bovine serum albumin as the background stain. This method was found to
be very useful for detecting organisms in samples that gave negative results by culture
(Brayton et al., 1986; Huq et al., 1990). It was later optimized and packaged as the
direct uorescent antibody staining kit for V. cholerae O1, Cholera DFA, by Hasan et al.
(1994). The IFA protocol remains useful for laboratories where commercial DFA kits for
V. cholerae are not readily available.
Materials
Concentrated water or homogenized plankton sample (Basic Protocol 2)
95% ethanol
FA Rhodamine counterstain (Becton Dickinson)
Polyvalent V. cholerae O1 antiserum (BD Difco)
FITC-conjugated anti-rabbit globulin goat serum (Sigma)
Mounting medium, such as FA (Difco) or Citiuor AF1/AF3 (Electron Microscopy
Sciences)
Multiwell Teon-coated slides
Incubators, set to 55
C and 35
C
Humid chamber: 50-ml centrifuge tube containing one to two strips of lter paper
saturated with deionized H
2
O
Glass coverslips
Epiuorescent microscope with FITC bandwidth lter
Detection of Vibrio
cholerae from the
Environment
6A.5.40
Prepare and x samples
1. Add an appropriate amount (dependent on concentration of sample and well size) of
each sample to be tested to a Teon-coated multiwell slide. Air dry (15 to 20 min)
at room temperature.
2. Fix the sample by adding 95% ethanol to each well containing sample. Air dry (5 to
10 min) at room temperature.
3. Heat slide 10 min in a 55
C incubator.
Slides may be stored up to 1 month at 70
C at this point.
Staining procedure
4. Rinse the slide(s) with 50 ml PBS and air dry (15 to 20 min).
5. While slide is drying, prepare humid chamber for use. Equilibrate chamber in 35
C
incubator (15 min).
6. To each dry sample well, add 1 to 2 drops of a 1:20 dilution of FA Rhodamine
Counterstain. Incubate in humid chamber 30 min at 35
C.
Minimize exposure to light from this step forward.
7. Rinse slide in PBS by gently ooding (50 ml), then soak in the same solution
10 min at room temperature. Remove and rinse again briey in PBS.
8. Allow slide to air dry (15 to 20 min).
9. Add 5 to 10 l of V. cholerae O1-specic antiserum. Incubate in humid chamber
30 min at 35
C.
10. Repeat steps 7 and 8. Use fresh PBS for washing.
11. Add 1 to 2 drops undiluted FITC-conjugated anti-rabbit globulin goat serum and
incubate in humid chamber 30 min at 35
C.
12. Repeat washing steps 7 and 8 using fresh PBS.
13. Mount each slide with a glass coverslip and a low uorescence, anti-quenching
mounting medium, such as Citiuor AF1.
14. Examine samples immediately using a epiuorescent microscope with a FITC band-
pass lter.
REAGENTS AND SOLUTIONS
Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see
APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.
Alkaline peptone water, 1
10 g peptone
10 g sodium chloride
Adjust volume to 1 liter with water
Adjust pH to 8.6 with NaOH
Autoclave to sterilize
Store up to 6 months at 2
to 8
C
Alkaline peptone water, 10
100 g peptone
continued
Nonenteric
Gamma
Proteobacteria
6A.5.41
Adjust pH to 8.6 with NaOH
to 8
C
Blot neutralization solution
0.5 M TrisCl, pH 7.2 (APPENDIX 2A)
1.5 M sodium chloride
to 8
C
Brain-heart infusion agar (BHI)
7.7 g calf brain (infusion from 200 g; BD Difco)
9.8 g beef heart (infusion from 250 g; BD Difco)
10 g proteose peptone (BD Difco)
2 g dextrose
5 g sodium chloride
2.5 g disodium phosphate
15 g agar (BD Difco)
For use in Alternate Protocol 1 supplement with:
1 g esculin (0.1% nal; BD Difco)
500 mg ferric chloride (anhydrous, 0.05% nal)
Allow to cool to 50
C
Prepare agar stabs by pouring into 4 to 5-ml sterile vials
Cell lysis buffer
0.5 N sodium hydroxide
Prepare fresh
CTAB/NaCl solution
Add 4 g NaCl to 80 ml water and dissolve. Slowly add 10 g of cetyltrimethylam-
monium bromide (CTAB) while heating and stirring at 65
C. Adjust volume to
100-ml with water. Store up to 6 months at room temperature.
Detection buffer
0.1 M TrisCl, pH 9.5 (APPENDIX 2A)
Store up to 1 month at room temperature
Fluorescein-labeled Vchomim1276 probe
5
-ACTTTGTGAGATTCGCTCCACCTCG-3
, with 5
uorescein label
Resuspend probe to a working concentration of 25 ng/l. Add probe to hybridization
solution base (see recipe) at a ratio of 32 l per 10 ml of solution. Store indenitely
at 4
C.
Formaldehyde solution, 4%
8 g paraformaldehyde powder
200 ml PBS (APPENDIX 2A)
Prepare fresh
continued
Detection of Vibrio
cholerae from the
Environment
6A.5.42
In a ventilated hood, heat PBS to 60
C and add paraformaldehyde powder. Stir

until dissolved. If some powder remains, slowly raise the pH with 1 N NaOH (nal
pH, 6.9). Filter sterilize.
Gelatin agar (GA)
4 g neopeptone
1 g yeast extract
5 g sodium chloride
15 g gelatin
15 g agar
Store up to 1 month at 2
to 8
C
Hybridization solution
0.9 M NaCl
20 mM TrisCl, pH 7.2 (APPENDIX 2A)
0.01% (w/v) SDS
35% (v/v) formamide
Hybridization solution base
Prepare the following in DEPC-treated water (APPENDIX 2A)
0. 9 M NaCl
50 mM sodium phosphate buffer, pH 8.0 (APPENDIX 2A)
5 mM EDTA
0.5% (w/v) SDS
Precipitation will occur upon standing at room temperature. Heat to 45
to 50
C to resus-
pend.
Maleic acid buffer
0.1 M maleic acid
Adjust pH to 7.5 with solid NaOH
Prepare fresh
Modied nutrient agar
3 g beef extract
5 g peptone
15 g agar
to 8
C
Pre-washing solution
Freshly prepare 3SSC(APPENDIX2A) containing 0.1%(w/v) SDS using RNase-free
ingredients and DEPC-treated water (APPENDIX 2A).
Proteinase K solution (40-g/ml)
Dissolve 4 mg proteinase K in 100 ml 1 SSC (APPENDIX 2A). Store up to 2 months
at 20
C
Nonenteric
Gamma
Proteobacteria
6A.5.43
Stringency wash solution I
2 SSC (APPENDIX 2A)
0.1% (w/v) SDS
Prepare fresh
Stringency wash solution II
0.5 SSC (APPENDIX 2A)
0.1% (w/v) SDS
Prepare fresh
Tellurite taurocholate gelatin agar (TTGA)
10 g tryptone
5 g sodium taurocholate
1 g sodium carbonate
30 g gelatin
15 g agar
Boil to completely dissolve ingredients
Final pH should be 8.5. If it is not, adjust with HCl.
Autoclave and add potassium tellurite to 1% (w/v) nal
to 8
C
Thiosulfate citrate bile salts sucrose (TCBS) agar
5 g yeast extract
10 g peptone
10 g sodium thiosulfate
10 g sodium citrate
8 g ox bile
20 g sucrose
1 g ferric citrate
0.04 g bromothymol blue
0.04 g thymol blue
14 g agar
Boil to completely dissolve
IMPORTANT NOTE: Do not autoclave.
This medium is also available commercially as a dehydrated powder from Oxoid (Remel)
Washing buffer
0.1 M maleic acid
0.3% (v/v) Tween 20
Adjust pH to 7.5 with solid NaOH
Prepare fresh
Washing buffer solution
0.9 M NaCl
20 mM TrisCl, pH 8 (APPENDIX 2A)
0.01% (w/v) SDS
C
Detection of Vibrio
cholerae from the
Environment
6A.5.44
Washing solution
Freshly prepare 1SSC(APPENDIX2A) containing 0.1%(w/v) SDS using RNase-free
ingredients and DEPC-treated water (APPENDIX 2A).
COMMENTARY
Background Information
Understanding the natural ecology of an in-
fectious agent outside of the human body is
essential to understanding the epidemiology
of the associated disease and especially nec-
essary in any attempt to prevent illness caused
by exposure to that pathogen. It is well es-
tablished that V. cholerae is autochthonous
to the aquatic environment globally and even
in regions where cholera is absent (Haley
et al., 2012). However, these environments are
highly heterogeneous and change over time. It
is also well established that seasonal uctua-
tion in environmental parameters is associated
with changes in environmental pathogen den-
sities and disease prevalence. It is, therefore,
necessary to understand the inuence of the
environment on presence and number of these
organisms. This requires a holistic approach
toward investigation of the microbial ecology
of an infectious disease. Temporal and spatial
studies of V. cholerae in water, planktonic or-
ganisms, sediment, and shellsh, coupled with
recording physical and chemical parameters
of the study environment (regardless of pres-
ence or absence of V. cholerae), allow inves-
tigators to estimate changes in the presence
or density of V. cholerae in a particular re-
gion. This information can be used to help
predict the public health safety of water bod-
ies or seafood, thereby reducing risk of illness
and loss of revenue from inaccurate prediction
of the presence of pathogens in water bod-
ies used for recreation or shellsh harvesting.
Although cholera is a public health threat for
developing nations more than for developed
nations, methods for V. cholerae discussed in
this chapter can be applied to other waterborne
pathogens with addition of methods specic to
the microorganism of interest.
As commented above, multiple approaches
should be taken to study the ecology of
pathogens in the environment. The coupling
of methods can overcome limitations inherent
in any one particular method and allow con-
vergence on the precise answer(s). However,
increase in the cost and time commitment
needs to be considered. An investigator should
rst determine the data or results needed, ap-
propriateness of available methods, and feasi-
bility of carrying out the procedures to achieve
the goal. If the microorganism of interest is
not detected, it is not appropriate to conclude
that the pathogen is absent from the study, but
rather that it was not detected by the methods
used. In this scenario, use of multiple methods
reduces not detected results. Furthermore,
parallel replication of assays will enhance ac-
curacy and statistical power of the results.
Critical Parameters and
Troubleshooting
Successful detection and isolation of
V. cholerae is dependent on both its presence
in the environment as well as the method used
for detection. V. cholerae undergoes the VBNC
state at prolonged low temperatures, which
will inuence whether culture of the bac-
terium will succeed when water temperatures
are low. Lack of growth on media is indica-
tive of V. cholerae in the VBNC state rather
than an absence of V. cholerae in the water
body of interest. In such instances, inclusion
of direct detection methods (DFA, IFA, FISH,
direct PCR) will provide more precise results
with respect to the presence of V. cholerae.
If such methods are used in conjunction with
culturing methods, then the methods should
be used throughout the entirety of the study,
instead of alternating methods based on the
prevailing environmental conditions.
The culturing methods presented in this unit
are the most commonly used culture-based
protocols for isolation of V. cholerae (Morris
et al., 1979; Rennels et al., 1980). Other meth-
ods have been described and may be suitable
for certain study environments. Alkaline bile
peptone water (Spira et al., 1981), Monsurs
tellurite taurocholate broth (Monsur, 1961),
and sodium-gelatin phosphate broth (Rennels
et al., 1980) have been described as enrich-
ment media that are effective for culturing
V. cholerae. A second enrichment step may be
used, but will add an additional 6 to 8 hr to an
already lengthy protocol. There is also the con-
cern that repeated passaging of cells will allow
for population and genotypic alterations to oc-
cur by favoring cells that grow more readily
in enrichment broths, allowing for mutations
to accumulate, i.e., natural mutations occur-
ring during cell division. The latter is a con-
cern in isolating cells whose genomes will be
Nonenteric
Gamma
Proteobacteria
6A.5.45
sequenced, as accumulation of SNPs and ge-
nomic rearrangements and possibly horizontal
transfer of mobile elements or loss of genomic
islands in the enrichment broth will yield inac-
curate genomic data about the cells in the en-
vironment. Such data may erroneously lead to
assumptions on virulence factors, phylogeny,
or origin of the strains. Essentially, culturing
steps should be minimized to obtain accurate
results.
As V. cholerae can grow at a wide range
of temperatures, several incubation tempera-
tures in APW and TCBS, TTGA, or CA can
be used for V. cholerae isolation. Incubating
at the lower range of recommended tempera-
tures (near 30
C) may enhance the growth of

environmentally stressed cells exposed to low
temperatures, but this may require longer incu-
bation times on solid media. Incubating media
at higher temperatures (37
C) may inhibit
the growth of some non-Vibrio species but may
also inhibit the growth of stressed V. cholerae
cells. If possible, incubate media in parallel at
multiple temperatures. Furthermore, stepwise
increase in incubation temperature, 25
C (1
to 2 hr) 30
C (1 to 2 hr) 35
C can be
used to sensitize the stressed cells to adapt and
grow.
We strongly advise that a subculturing step
onto nonselective media be utilized after iso-
lation from TCBS, TTGA, or CA. This step
serves two purposes. First, it is critical that
pure colonies be isolated before proceeding to
any genomic or phenotypic characterization
steps. Secondly, growth on TCBS is not suit-
able for the oxidase test, serotyping, or direct
PCR.
It is known that PCRamplication of target
DNA in environmental samples can be inhib-
ited by dissolved organics, such as humic acid.
For that reason, we suggest performing a DNA
extraction rst. This typically adds only 3 to
4 hr to the protocol and results in high-yield
DNAwith a decrease in inhibition during PCR
amplication. For all direct PCRexaminations
(PCRon extracted or boiled APW), include the
eubacterial PCR reaction to conrm template
quality on samples. Further, we strongly sug-
gest that direct PCR be done on 1:10, 1:100
dilutions (DNA template:TE buffer or DNA
template:nuclease free water) in parallel with
undiluted samples.
FISH allows visual quantication of all
V. cholerae cells, regardless of their cultur-
ability, in a sample. One problem with this
method is observation of autouorescing con-
stituents found in environmental water sam-
ples. All positive samples should be doc-
umented by digital or lm photography, and
conrmation by conventional PCRand/or real-
time PCR of a parallel sample (frozen, but not
xed) is advisable.
Table 6A.5.6 outlines some of the more
common problems that may be experienced in
performing the basic and alternate protocols
from this unit. This is not an exhaustive list;
others may be encountered. Please consult the
troubleshooting sections from the referenced
units of Current Protocols in Molecular Biol-
ogy (see Literature Cited) for further advice.
Anticipated Results
V. cholerae can be easily isolated fromestu-
arine environments during the warm summer
months, even in non-epidemic areas. Chances
for successful isolation decrease during the
colder winter months, even from samples that
are positive by PCR or DFA. Figure 6A.5.3
shows the typical growth of V. cholerae on
TCBS, TTGA, and CA media. Growth on
TCBS by V. cholerae appears as small (1 to
3-mm diameter), at, yellow colonies (Fig.
6A.5.3). V. cholerae appear as mediumto large
(2 to 5-mmdiameter), at, translucent colonies
on TTGA (Fig. 6A.5.3). There will be a zone
of clearing (or halo) surrounding V. cholerae
colonies due to hydrolysis of gelatin. A dark
center usually develops after 24 hr. Growth on
CA by V. cholerae appears as medium to large
(2- to 5-mm diameter) turquoise colonies.
However, V. cholerae is a highly heteroge-
neous species and can exhibit various colony
morphologies including a rugose form(White,
1940). Several different V. cholerae isolates
should be used as positive controls to be com-
parators for cultured cells. TCBS, TTGA, and
CA are all selective, but still allow the growth
of other Vibrio species and related bacteria.
For example, V. parahaemolyticus will appear
as blue-green (occasionally yellow) colonies,
V. vulnicus will appear as greenish yellow
colonies, and V. alginolyticus as larger, some-
times swarming yellow colonies on TCBS.
Therefore, colonies growing on these media
are not necessarily all V. cholerae; hence, they
are labeled as presumptive until they can be
conrmed by PCR. If several of these solid
media are available to the investigator, then it
may be benecial to dot or streak for isola-
tion presumptive V. cholerae colonies isolated
from one medium onto another that is selec-
tive for V. cholerae as well (TCBS CA
and TTGA, for instance). Visualizing the color
and morphology of an isolate on one selective
medium that was recovered from a different
selective medium may help the investigator
Detection of Vibrio
cholerae from the
Environment
6A.5.46
Table 6A.5.6 Common Problems that May be Experienced in Detection, Isolation, and Identication of V. cholerae
from the Environment
Problem Possible solution(s)
Basic Protocol 1
Little or no plankton in cod-end
collecting bucket
Check pore size of plankton net and ensure that it is 64 mm
Zooplankton should be sampled near dawn or dusk (1-2 hr after sunrise or
before sunset) when they are nearer to the surface.
Filter more water through plankton net
Basic Protocol 2
No V. cholerae growth Adjust dilution volumes and/or incubation temperature
Incubate petri dishes at several temperatures
V. cholerae overgrowth Adjust dilutions or use a smaller volume loop to spread onto agar
Basic Protocol 3
Autoagglutination or clumping in saline
without antisera
Rough morphotypes cannot be serogrouped with antisera. Use O1/O139
rfb PCR primers with Basic Protocol 4 to test for toxigenic serogroups.
Support Protocol 1
Low DNA concentration Increase volume of boiled cells
Basic Protocol 4
No PCR product with positive control Ensure all components are added to reaction at the proper concentration
Use fresh dNTPs
Prepare fresh crude template of positive control as it will degrade over time
Dilute crude template 1:5000 or more and repeat the control reaction
Quantify crude template by gel electrophoresis (10 ml) to ensure
sufcient template concentration
PCR product with negative control Most likely caused by carry-over contamination in one of the reaction
components. Make new components.
Basic Protocols 6 and 7
Weak amplication with positive control Ensure all components are added to reaction at the proper concentration
Use fresh dNTPs
Strong amplication with negative
control
Most likely caused by carry-over contamination in one of the reaction
Basic Protocol 9
Positive control is negative Ensure solutions used are RNase-free
Increase incubation time of lysis step (10% SDS), especially if colonies are
larger than 3 mm
Ensure that the correct microscope lter is used for uorochrome selected.
Other uorochromes may be used.
continued
Nonenteric
Gamma
Proteobacteria
6A.5.47
Table 6A.5.6 Common Problems that May be Experienced in Detection, Isolation, and Identication of V. cholerae
from the Environment, continued
Problem Possible solution(s)
Positive control gives weak signal Check scanning settings on detection instrument
Increase probe concentration and/or hybridization time
Alternate Protocol 3
Positive control is negative or weak Ensure that solutions are used in correct order
Overexposure to UV source will degrade DNA template. Consider using
positively charged nylon membranes, which do not need cross-linking.
Extend hybridization time
Extend development time
Positive control is overdeveloped or
background is high
Check hybridization temperature
Do not allow membrane to dry
Decrease development time
Positive control is negative or weak Check efciency of probe labeling reaction
Increase probe concentration ( 25 ng/ml)
Basic Protocol 10
Weak uorescence Increase probe concentration
Check hybridization temperature
Increase hybridization time
Nonspecic staining Perform more stringent wash step
Basic Protocol 11
No PCR product with positive control Ensure all components are added to reaction at the proper concentration
Use fresh dNTPs
PCR product with negative control Most likely caused by carry-over contamination in one of the reaction
Positive control is negative Ensure that the proper lter set is used on the uorescent microscope
Bengal DFA (V. cholerae O139) kit positive control is sometimes poor.
Prepare positive control from laboratory reference strain.
Basic Protocol 12
Positive control signal is weak Increase amount of V. cholerae O1 antiserum and FITC conjugate
Detection of Vibrio
cholerae from the
Environment
6A.5.48
determine whether or not to proceed with PCR
conrmation of that isolate.
Table 6A.5.4 lists a comprehensive, yet not
exhaustive set of PCR primers used to charac-
terize V. cholerae isolates. This table also lists
the reaction temperatures and times as well as
the expected amplicon size. Positive control
strains known to encode the target region and
yielding an appropriate size amplicon should
be used for all PCR reactions.
Time Considerations
Choosing the appropriate methods dis-
cussed in this unit will determine how rapidly
results can be achieved. Clinical diagnosis
requires rapid determination of the etiologi-
cal agent, and rapid non-culturing techniques
are currently being developed. In the case of
cholera, symptoms are often distinguishable
from other infections, and stool typically re-
quires no enrichment in APW and readily
yields V. cholerae colonies on TCBS agar. En-
vironmental monitoring often requires more
time, depending on the method. However, PCR
screening can be performed in 6 to 24 hr, de-
pending on the template, and DFAcan be done
in 4 hr or 24 hr if coupled with DVC. Use of
multiple methods and genotypic characteriza-
tion will signicantly increase the amount of
time needed to complete this work. In-depth
genotypic characterization can be done in in-
tervals after several rounds of sample collec-
tion and V. cholerae isolation. It is important
to conrm any presumptive strain as being
V. cholerae before time and resources are ded-
icated to genotypic characterization. As with
any proposed work, a daily schedule with esti-
mated timeframes may be helpful in efciently
accomplishing the investigators goals.
Acknowledgements
This work was partially supported by Na-
tional Science Foundation Grant 0813066,
NIH Grant 2RO1A1039129-11A2, National
Institutes of Health-Fogarty International Cen-
ter Grant 1RC1TW008587-01, U.S. Army
Medical Research and Material Com-
mand Grant W81XWHO9201, NOAA Grant
S0660009, and DHS Contract HSHQDC-10-
C-00177.
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UNIT 6A.

5 Detection, Isolation, and Identication of

C. Stock cultures of V. cholerae

C until processing begins (not to exceed 8 hr;

C for 15 min. Oysters can be shucked when the blade is cool.

C for 24 2 hr and TTGA plates at 30

C for TCBS and CA or 48 hr at 30

C for 2 hr. Sterilization can also be

C for 1 to 2 hr. If it is not possible to procure

C overnight (or 48 hr if TTGA agar is

C for 72 hr, or at room temperature

reporter dye (FAM-6-carbooxyuorescein) and a 3

C water bath or heat block

C. Wash DNA with 1 ml of

(Heidelberg, 1997; Heidelberg

C and then proceed to step 7.

C on fresh lter paper wetted with 3 SSC.

C in hybridization solution base at a ratio

C. Wrap the master plate with Paralm and keep at 10

C with 100 ml proteinase K solution using gentle

C in antibody solution (1:10,000 dilution of

C for 24 hr in the humid chamber using hybridization

C for 10 min before washing the slides

C) a parallel sample (1 ml) for PCR conrmation.

C for a minimum of 6 hr to overnight.

C in the dark for up to 6 months.

C in humid chamber. Protect slide from light.

C and add paraformaldehyde powder. Stir

C) may enhance the growth of

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